KR20040015013A - Stimulation of Thymus for Vaccination Development - Google Patents

Abstract

The present invention provides a method for improving the response of the patient's immune system to inoculation. This is accomplished by reactivating the thymus. Optionally, self-derived or genetic syngeneic, allogeneic, or heterologous hematopoietic hematopoietic stem cells are delivered to the patient's immune system to accelerate the rate of regeneration. In a preferred embodiment the hematopoietic stem cells are CD34 ⁺ . The thymus of the patient is reactivated by disrupting signaling to the thymus mediated by sex steroids. In a preferred embodiment, the disruption is provided by administering an LHRH agonist, an LHRH antagonist, an anti-LHRH receptor antigen, an anti-LHRH vaccine or a combination thereof.

Description

Stimulation of Thymus for Vaccination Development

Immune system

The main function of the immune system is to distinguish the "foreign" antigens from "self" and to respond accordingly to protect the body from infection. This definition may also be described as distinguishing a "bad" molecule from a "good" molecule. In a normal immune response, the antigenic expression of a specific purpose, which captures foreign antigens and processes them into small peptide fragments, expressing the fragments in the cleft of major histocompatibility complex (MHC) molecules on the surface of antigen-expressing cells (APC). Cells are involved in sequential events. MHC molecules may be Class I (recognized by cytotoxic T lymphocytes (Tc)) expressed in all nucleated cells or Class II (recognized by helper T lymphocytes (Th)) expressed primarily by cells of the immune system.

APC settlement is programmed for antigen capture and processing into peptide fragments and then expressed on surface MHC molecules. This more activated APC can then stimulate T cells better than to capture and process additional antigens. Th cells recognize and respond to MHC II / peptide complexes on APC.

There are two types of Th cells that are distinguished by different types of soluble regulatory factors that the two types of Th cells produce. Th1 cells mainly produce IL2 and gamma interferon (IFNγ). The latter promotes preferential activation of cell mediated immunity (primarily Tc) when present at the time of initial contact of naïve T cells with the antigen. Th2 cells express different profiles of cytokines, especially IL4, 5 and 10, which induce humoral immunity through antibodies to produce B cells specific for specific antigens. In some cases, this can result in inappropriate allergic reactions through IgE production.

In practice, the importance of Th cells in all immune responses is best seen in HIV / AIDS, and the absence of Th cells through destruction by the virus results in severe immune deficiency and eventually death. In the central importance of Th cells as immunoregulatory cells, the balance between Th1 and Th2 cells can severely affect the nature of the immune response. Such imbalance can occur through developmental dysplasia or improper activation at the beginning of an immune response. This can lead to various diseases such as allergies, cancer and autoimmunity.

Thymus

Because the thymus is the primary site where T lymphocytes are produced, the thymus is arguably the main organ in the immune system. The action of the thymus is to attract appropriate bone marrow-derived precursor cells from the blood to induce the consignment of the precursors to the T cell lineage, including gene rearrangements essential for the production of T cell receptors (TCRs) for antigen. Related to this is an increase in the number of T cells through an excellent degree of cell division, increasing the likelihood that all foreign antigens will be recognized and eliminated. This huge potential diversity means that a large number of lymphocytes will be able to respond to varying strengths (affinity) of any single antigen to which the body can come into contact. However, a unique feature of antigen recognition by T cells is that, unlike B cells, TCR only recognizes peptide fragments that are physically bound to MHC molecules, commonly referred to as autologous MHC, and this ability is selected in the thymus. This process is called positive selection and is a proprietary feature of cortical epithelial cells. If the TCR fails to bind to the autologous MHC / peptide complex, the T cells are killed by "neglect"-this requires some signal transduction through the TCR for continued maturation.

Upon selection in the cortex, developing thymic cells require functional maturation and mobile capacity and are released into the blood stream as native (not yet contacted antigen) T cells. The thymic cells circulate in search of antigen between lymph and blood. If thymic cells are not stimulated after 3-4 weeks, thymic cells are prone to deletion from the peripheral T cell population by other recent thymic effluents. This system of thymic export and peripheral T cell substitution provides a continuous complement of T cell quality with homeostasis maintaining adequate levels.

The thymus releases about 1% of its T cell content into the blood per day as a basis for the functional immune system, but one of the obvious variants of mammals is that the organ develops severe atrophy as a result of the production of sex steroids. will be. It can also begin in children, but it gets worse in puberty. In normal healthy individuals, this loss of production and release of new T cells do not always have clinical significance. In fact, the thymus of old age is atrophic and consists of less than 1% of its young counterparts (see below), but still continues to release very low levels of new T cells into the blood stream. They are insufficient to maintain optimal levels of peripheral T cell subsets, but raise the possibility that the thymus is not completely dominant so that the thymus may be the target of therapy.

With progressive aging, decline in thymus export means that the state of peripheral T cells undergoes progressive changes both quantitatively and qualitatively. In addition to the gradual decrease in absolute T cell numbers in the blood as they age, the relevant antigen specific native T cells (those that have not yet experienced the antigen) are stimulated and multiply because they die through the lack of stimulation with each antigen contact. The subset will proceed to effector cells to remove pathogens, but they eventually die through antigen induced cell death.

Another subset will be converted to memory cells to provide long term protection against future contact with the pathogen. Thus, there is a decrease in the level of naïve T cells and as a result the ability to respond to antigen is reduced. Aging also results in selective decay in Th cells (characterized by the expression of CD4) and imbalance of the ratio of Th1 to Th2 cells to Tc cells (expressing CD8). This does not occur in normal young, as there is a continuous supply of new T cells exported from the thymus as mentioned above, thus providing a continuous supplementation of the native T cell population at the periphery.

Thymic atrophy

Thymus is affected to a significant extent by two-way communication with the neuroendocrine system (Kendall, 1988). Of particular importance is thyroid functioning including trophic effects [thyroid stimulating hormone (TSH) and growth hormone (GH)] and atrophic effects [luteal hormone (LH), follicle stimulating hormone (FSH) and corticosteroids (ACTH)]. It is an interaction between the pituitary gland, the adrenal gland and the gonads [Kendall, 1988; Homo-Delarche, 1991]. One of the unique physiological features of the thymus is the gradual degeneration of structure and function, which is equivalent to an increase in the production of circulating sex steroids near puberty [Hirokawa and Makinodan, 1975; Tosi et al., 1982; Hirokawa, et al., 1994]. The exact targets of the hormones and mechanisms that induce thymic atrophy are not yet known. Since the thymus is the primary site for the generation and maintenance of peripheral T cell pools, thymic atrophy has been widely recognized as a major cause of the increased incidence of immune system disorders with older age. In particular, immune system deficiency, exemplified by a decrease in T cell dependent immune function, such as cytotoxic T cell activity and mitotic promoter responses, results in increased incidence of immunodeficiency, autoimmunity and tumors later in life [Hirokawa, 1998]. .

The effect of thymic atrophy is seen at the periphery, where thymic signal input to T cell populations decreases the diversity of T cell receptor (TCR) repertoires. Altered cytokine profiles [Hobbs et al., 1993; Kurashima et al., 1995], changes in CD4 ⁺ and CD8 ⁺ subsets, and bias toward memory T cells for naive T cells (Mackall et al., 1995) are also observed. Moreover, the efficiency of thymus production is impaired with age so that after the T cells are depleted, the immune system's ability to regenerate normal T cell numbers is eventually lost (Mackall et al., 1995). However, recent studies by Dou et al. Have shown that thymic signal output may occur in older humans [Douek et al. (1998). The DNA cleavage product of the TCR-gene rearrangement was used to demonstrate the presence of newly generated native T cells circulating after HIV infection in elderly patients. The signal output and then the peripheral reproduction ratio of T cell population is, in the case of the patients treated with chemotherapy compared to patients before puberty as the reproduction rate of the T cell population (in particular, CD4 ⁺ T cells) dramatically in patients after puberty It has been reduced and therefore needs further study (Mackall et al., 1995). This is further exemplified in a recent study by Tim and Thoman (1999), which showed poor thymic production of native T cells, even if CD4 ⁺ T cells are regenerated in older mice after bone marrow transplantation (BMT). It has been demonstrated that due to the peripheral microenvironment of the old age, it is biased into memory cells.

The thymus consists essentially of developing thymic cells, which are scattered within various interstitial cells (mostly epithelial cell subsets) that make up the microenvironment and provide the growth factors and cellular interactions necessary for optimal development of T cells. have. The symbiotic developmental relationship between epithelial cell subsets and thymus that regulates thymus differentiation and maturation [Boyd et al., 1993] suggests that steroid suppression may affect the state of one cell type (and later other cell types). May occur at the level of). In previous studies with radiation chimeras, bone marrow (BM) hepatocytes were not affected by age [Hirokawa, 1998; Mackall and Gress, 1997] Since it has been shown to have similar thymic reconstitution capacity as young BM cells, the chance of inherent defects within the thymus cells itself is low. Moreover, thymic cells of old animals have at least some degree of differentiation [Mackall and Gress, 1997; George and Ritter, 1996; Hirokawa et al., 1994]. However, a recent study by aspinal [Aspinall (1997)] demonstrated a defect in the CD3 ^- CD4 ^- CD8 ^- triple negative (TN) population that occurs during the TCRγ chain gene-rearrangement step.

Aging is not only a condition that results in T cell loss, but it also occurs very seriously after chemotherapy or radiotherapy, for example in HIV / AIDS. In addition, the recovery of the immune system (via T cell-mediated immunity) in young children with active thymus occurs relatively quickly (2 to 3 months) compared to after puberty if it can take 1 to 2 years due to atrophic thymus. .

vaccine

Vaccines can be divided into two classes: providing active vaccinations and providing passive vaccinations. Passive vaccination involves administering an antibody from a heterologous source to a patient in response to the patient's foreign antigen or antigen that the patient may experience. Such vaccination is usually very temporary because the patient's original immune system is not involved. The present disclosure is in the field of active vaccination in which the antigen is administered to the patient and then the patient's immune system responds to the antigen by forming an antibody specific for the antigen.

There are several parameters that can affect the nature and extent of the immune response: the level and type of antigen, the site of vaccination, the availability of appropriate APCs, the general health of the individual and the state of the T and B cell population. Of these, T cells are most vulnerable because of the significant sex steroid-induced arrest in thymic exports that aggravate from the onset of puberty. Thus, any vaccination program is logical only if the level of potential responder T cells is optimal for both the presence of native T cells exhibiting the specificity of a broad repertoire, and the normal ratio of Th1 to Th2 cells and Th to Tc cells. Should be undertaken. In addition, the levels and types of cytokines must be engineered to suit the desired reaction.

The ability to reactivate atrophic thymus via inhibition of sex steroid production, for example, at the signaling level of luteinizing hormone secretory hormone (LHRH) to the pituitary gland, has the potential to produce new herds of native T cells and TCR types of various repertoires. Provide a viable means of generating. This process does so by using normal regulatory molecules and pathways that effectively return the thymus to its pre-pubertal state and produce optimal thymus production.

Summary of the Invention

The present disclosure relates to a method of improving the immune response of an animal against a vaccine. This is achieved by restoring the peripheral T cell population quantitatively and qualitatively, especially at the level of native T cells. These native T cells can then respond to a greater extent to the foreign antigen provided.

The method of the invention relies on blocking signal transduction to the thymus mediated by sex steroids. In a particularly preferred embodiment, chemical castration is used. In another embodiment surgical castration is used. Castration reactivates the thymus by returning the state of the thymus to its pre-pubertal state.

In certain embodiments, signaling to the thymus mediated by sex steroids may be agonists or antagonists of LHRH, anti-estrogen antibodies, anti-androgen antibodies, or passive (antibody) or active (antigen) anti-LHRH vaccines or these Is blocked by the administration of a combination of ("blockers").

In a preferred embodiment, the blocker (s) is administered as a sustained release peptide formulation. An example of a sustained release peptide-releasing agent is provided in WO 98/08533, which is incorporated herein by reference in its entirety.

The present disclosure belongs to the field of response to vaccines in animals. Specifically, the present invention belongs to the field of improving vaccine response through stimulation of the thymus.

1A and B: Changes in thymic cell number before and after castration. With age, thymic atrophy significantly reduced the number of thymic cells. After two weeks of castration, the cell number increased to the level of young mature mice. After 3 weeks of castration, there was a significant increase in the level of young mature mice and stabilized after 4 weeks of castration. *** = significantly different from thymus in young mature mice (2 months) (p <0.001)

Figures 2A-C: (A) The number of cells in the spleen remained constant regardless of age and remained constant even after castration. Peripheral B cell to T cell ratios also remained constant (B), but the ratio of CD4 to CD8 decreased significantly with age (p <0.001) and returned to normal young mouse levels after 4 weeks of castration.

Figure 3: Fluorescence activated cell sorting (FACS) profile for CD4 versus CD8 thymic cell population after age and castration. The percentage of each quadrant is shown at the top of each plot. The subpopulation of thymic cells remained constant regardless of age, and after castration there was a simultaneous increase in thymic cells.

4: Proliferation rate (%) of thymic cells detected by the amount of BrdU pulse incorporation. The rate of proliferation of thymic cells remains constant regardless of age, even after castration.

5A-D: Effect of age and castration on proliferation of thymic cell subsets. (A) Proportion of each subset making up the entire proliferating population-The proportion of CD8 ⁺ T cells in the proliferating population was significantly increased. (B) Percentage of each subpopulation proliferating—TN and CD8 subsets significantly less proliferated in 2 year old mice than in 2 month old mice. After two weeks of castration, the proliferation rate of the TN population recovered to normal young mouse levels, but the proliferation rate of the CD8 population was found to increase significantly. After 4 weeks of castration, the levels were equivalent to normal young mouse levels. (C) The overall TN proliferation rate remains constant regardless of age, and remains constant even after castration. However, (D) significant decrease in TN1 subpopulation growth with age did not return to normal levels after 4 weeks of castration. *** = very significant (p <0.01) ** = significant (p <0.01)

Figure 6: Mice were injected intrathymally with FITC. After 24 hours the number of peripheral FITC ⁺ cells was counted. The percentage of percent thymic migrants (RTE) remained steady at about 1% of thymic cell numbers with age, but significantly decreased after two weeks of castration, with a significant decrease in the number of RTE cells with age (p <0.01). After castration, these numbers increased, but were significantly lower than the young mice even in the second week after castration. With age, the ratio of CD4 ⁺ RTE to CD8 ⁺ RTE was found to increase significantly, which normalized after 1 week of castration.

7A-C: Changes in thymus (A), spleen (B) and lymph node (C) cell numbers after treatment with cyclophosphamide, a chemotherapeutic agent. It should be noted that in the thymus of animals castrated at weeks 1 and 2 post-treatment, the cell number increased rapidly compared to the group that did not castrate (treated with cyclophosphamide only). In addition, the number of spleen and lymph nodes in the castrated group also increased significantly compared to the group treated with cyclophosphamide alone. After 4 weeks the cell number normalized (n = 3-4 animals at each treatment group and each time point).

8A-C: Changes in thymus (A), spleen (B) and lymph node (C) cell numbers after irradiation (625 Rads) one week after surgical castration. It should be noted that at weeks 1 and 2 post-treatment, the number of cells increased rapidly in the thymus of castrated animals compared to the non-castrated (only irradiated) group (n = 3 to each treatment group and each time point). 4 horses).

9A-C: Changes in thymus (A), spleen (B) and lymph node (C) cell numbers after irradiation and castration with the same. At week 2 post-treatment, it should be noted that in the thymus of castrated animals there was a rapid increase in cell numbers compared to the non-castrated groups. However, when compared to mice castrated 1 week prior to treatment (FIG. 7), no apparent difference was observed (n = 3-4 mice at each treatment group and each time point).

Fig. 10: Changes in thymus (A), spleen (B) and lymph node (C) cell numbers after performing castration or chemical castration by the chemotherapeutic cyclophosphamide treatment and surgery with the same. At weeks 1 and 2 post treatment, it should be noted that in the thymus of castrated animals there was a rapid increase in cell numbers compared to the group that did not castrate (treated with cyclophosphamide only). In addition, in the spleen and lymph nodes of the castrated group, the number of cells increased significantly compared to the group treated with cyclophosphamide alone (n = 3 to 4 mice at each treatment group and each time point). With regard to immune system regeneration following cyclophosphamide treatment, chemical castration was comparable to surgical castration.

Figure 11: Cellularity of lymph nodes after plantar immunization with herpes simplex virus-1 (HSV-1). It should be noted that after age castration, the cell fidelity was increased in the mice that were not castrated older mice. The lower graph illustrates the total activated cell count gated against CD25 vs CD8 cells via FACS.

12A-C: Vβ10 expression in CTLs (cytotoxic T lymphocytes) in activated LNs (lymph nodes) after HSV-1 inoculation. It should be noted that reduction in clone response and recovery of reactor replacement after castration in older mice.

13A-C: Recovery of responsiveness to HSV-1 immunization by castration. (a) After infection, older mice were found to significantly reduce overall lymph node cell fidelity compared to young mice and mice of same age after castration. (b) Representative FACS profiles of (CD8 ⁺ CD25 ⁺ ) cells activated in LN of mice infected with HSV-1. No difference in the proportion of CTLs activated with age or after castration was observed. (c) The decrease in cellular integrity in lymph nodes of older mice was reflected by a significant decrease in the number of activated CTLs. When old mice were castrated, the immune response to HSV-1 was restored, which was equivalent to that of young mice with CTL counts. Results are shown as mean ± 1SD of 8-12 mice. ** = p ≦ 0.01, compared to young mice (2 months); ^ = p ≤0.01, compared to older (no castration) mice.

14: The supine lymph nodes were dissected and cultured for 3 days in mice immunized with HSV-1. CTL assays were performed (measured with ⁵¹ Cr-release) using unimmunized mice as controls for background levels of lysis rate. The results are shown as the mean ± 1 SD of three mice. Older mice showed a significant (p ≦ 0.01, *) decrease in CTL activity at both E: T ratios of 10: 1 and 3: 1, indicating the percentage of specific CTL present in lymph nodes (% ) Indicates a decrease. When castrated older mice, the CTL response was restored to the level of young mature mice.

15A and B: Analysis of CD4 ⁺ T helper cells and VβTCR response to HSV-1 infection. On day 5 after HSV-1 infection, the supine lymph node was dissected (a) CD25, CD8 and specific TCRVβ markers. And (b) in vitro analysis for expression of CD4 / CD8 T cells. (a) The percentage (%) of activated (CD25 ⁺ ) CD8 ⁺ T cells expressing Vβ10 or Vβ8.1 is expressed as mean ± 1SD of 8 mice per group. Depending on age or after castration, no difference was observed. (b) The CD4 / CD8 ratio in the uninfected LN population was observed to decrease with age. It recovered after castration. The results are shown as mean ± 1SD of 8 mice per group. *** = p ≦ 0.001, compared to young and castrated mice.

16A-D: Changes in thymus (A), spleen (B), lymph node (C) and bone marrow (D) cell numbers after bone marrow transplantation in Ly5 congenic mice. It should be noted that after treatment, the thymus cell number increased rapidly at all time points in the castrated animals compared to the non-castrated groups. In addition, the number of spleen and lymph nodes increased significantly in the castrated group compared to the group treated with cyclophosphamide alone (n = 3-4 at each treatment group and each time point). In castrated mice there was a significant increase in pseudogenic (Ly5.2) cells compared to non-castrated animals (data not shown).

17A and B: Changes in thymic cell numbers in castrated and non-castrated mice following fetal liver reconstitution (n = 3-4 per test group). (A) At week 2, the thymic cell counts of castrated mice were normal and significantly higher than the mice that were not castrated (* p ≦ 0.05). After 4 weeks, no hypertrophy was observed in the thymus of castrated mice. The cell number of uncastrated mice remained below control levels. (B) CD45.2 ⁺ cells-CD45.2 ⁺ is a marker indicating that the donor is derived. Two weeks after reconstitution, donor-derived cells were present in both castrated and non-castrated mice. Four weeks after treatment, about 85% of cells in castrated mouse thymus were from donors. There was no donor-derived cell in the thymus uncastrated.

18: FACS profile of thymic cell populations from CD4 vs CD8 donors following surgical castration following lethal irradiation and fetal liver reconstitution. The percentage (%) of each quadrant is shown to the right of each plot. The control profile of the same age was the thymus of 8 month old Ly5.1 pseudogenic mice. Castrated and non-castrated mice were gated against CD45.2 ⁺ cells, showing only donor derived cells. Two weeks after reconstitution, thymic cell subpopulations did not differ between castrated and uncastrated mice.

19A and B: Myeloid and lymphoid dendritic cell (DC) numbers (n = 3-4 mice per test group) after lethal irradiation, fetal liver reconstitution and castration. In the graph, the control (white bar) is the normal number of dendrites present in untreated mice of the same age. (A) Myeloid dendrites from donor-After 2 weeks of reconstitution, DCs were present at normal levels in mice that were not castrated. At the same time point, the number of DCs in castrated mice was significantly (* p ≦ 0.05) higher. At week 4, DC numbers in castrated mice remained above control levels. (B) Lymphoid dendritic cells from donor—After two weeks of reconstitution, the number of DCs of castrated mice was twice that of non-castrated mice. After 4 weeks of treatment, DC counts remained above control levels.

20A and B: Changes in total number of cells and CD45.2 ⁺ bone marrow cell numbers in castrated and non-castrated mice following fetal liver reconstitution (n = 3-4 mice in each test group). (A) Total Number of Cells-Two weeks after reconstitution, myeloid cell numbers normalized and there was no significant difference between castrated and non-castrated mice. After 4 weeks of reconstitution, there was a significant difference in cell numbers between castrated and uncastrated mice (* p ≦ 0.05). (B) CD45.2 ⁺ cell number. After 2 weeks of reconstitution, there was no significant difference in the number of CD45.2 ⁺ cells in bone marrow between castrated and non-castrated mice. At week 4, CD45.2 ⁺ cell numbers remained maintained at high levels in castrated mice. At the same time point, the mice that were not castrated had no donor-derived cells.

21A-C: Changes in T cell number and number of dendrites (DC) derived from bone marrow and lymphoid bone marrow in castrated and non-castrated mice following fetal liver reconstitution (n = mouse 3 for each test group) To 4). In the graph, the control (white bar) is the normal number of T cells and dendrites present in untreated mice of the same age. (A) T cell numbers—After 2 and 4 weeks of reconstitution, T cell numbers decreased in both castrated and uncastrated mice. (B) Myeloid dendrites from donor-After 2 weeks of reconstitution, DC cell numbers were normal in both castrated and uncastrated mice. At this point, there was no significant difference in cell numbers between castrated and uncast casted mice. (C) Lymphoid dendrites from donor-2 and 4 weeks after reconstitution, the cell number was normal. At week 2 there was no significant difference in this figure between the castrated and non-castrated mice.

22A and B: Changes in total number of cells and donor (CD45.2 ⁺ ) splenic cell numbers in castrated and non-castrated mice following fetal liver reconstitution (n = 3-4 mice in each test group). (A) Total Number of Cells-Two weeks after reconstitution, the cell number was reduced and there was no significant difference in cell number between the castrated and non-castrated mice. Four weeks after reconstitution, the cell counts of castrated mice reached normal levels. (B) CD45.2 ⁺ cell numbers—After two weeks of reconstitution, there was no significant difference in the splenic CD45.2 ⁺ cell numbers between castrated and uncastrated mice. At week 4, CD45.2 ⁺ cell numbers remained maintained at high levels in castrated mice. At the same time point, the mice that were not castrated had no donor-derived cells.

23A-C: After fetal liver reconstitution, splenic T cells and dendritic cells (DC) derived from myeloid and lymphoid (n = 3-4 mice for each test group). In the graph, the control (white bar) is the normal number of T cells and dendrites present in untreated mice of the same age. (A) T Cell Number—After 2 and 4 weeks of reconstitution, T cell numbers decreased in both castrated and non-castrated mice. (B) (CD45.2 ⁺ ) myeloid dendrites from donor-After 2 and 4 weeks of reconstitution, DC numbers were normal in both castrated and non-castrated mice. At week 2, there was no significant difference between castrated and non-castrated mice. (C) (CD45.2 ⁺ ) lymphoid dendrites from donor-2 and 4 weeks after reconstitution, the cell number was normal. At week 2 there was no significant difference in this figure between the castrated and non-castrated mice.

Figures 24A and B: Changes in total number of cells and donor (CD45.2 ⁺ ) lymph node cell numbers in castrated and non-castrated mice following fetal liver reconstitution (n = 3 to 4 mice for each test group) ( A) Total Number of Cells-Two weeks after reconstitution, the cell number was normal and there was no significant difference between the castrated and non-castrated mice. After 4 weeks of reconstitution, the number of cells in castrated mice was normal. (B) CD45.2 ⁺ cell number—After two weeks of reconstitution, there was no significant difference in the number of donor CD45.2 ⁺ cells in lymph nodes between castrated and non-castrated mice. At week 4, CD45.2 cell counts remained high at the castrated mice. At the same time point, the mice that were not castrated had no donor-derived cells.

25 A-C: Changes in T cells and myeloid and lymphoid dendritic cells in mesenteric lymph nodes of castrated and uncastrated mice following fetal liver reconstitution (n = 3-4 mice per test group). The control (white bar) is the number of T cells and dendrites present in untreated mice of the same age. (A) After 2 and 4 weeks of reconstitution, T cell numbers were reduced in both castrated and non-castrated mice. (B) Myeloid dendrites from donor were normal in both castrated and uncastrated mice. After 4 weeks, the cell count decreased. At week 2, there was no significant difference in cell count between castrated and uncastrated mice. (C) Lymphoid dendrites from donor-Cell counts at 2 and 4 weeks after reconstitution were normal. At week 2, there were no significant differences in the cell numbers of castrated and non-castrated mice.

FIG. 26: Characterization of peripheral blood lymphocytes was analyzed in human patients (all over 60 years old) who underwent LHRH-agonist treatment for prostate cancer. Patient samples were analyzed before treatment and 4 months after LHRH-agonist treatment. The total cell count of lymphocytes per ml of blood was the lowest of the control values prior to treatment in all patients. After treatment, total lymphocyte counts were found to increase substantially in 6 of 9 patients (in some cases a 2 fold increase in total cells was observed). In this regard, total T cell numbers increased in 6 of 9 patients. Within the CD4 ⁺ subset, this increase is even more pronounced, with an increase seen in 8 of 9 patients, demonstrating an increased level of CD4 T cells. This trend was not evident within the CD8 ⁺ subset and was generally seen to increase in 4 out of 9 patients, although less than CD4 ⁺ T cells.

27: Analysis of human patient blood before and after LHRH-agonist treatment demonstrated no substantial change in the overall ratio of T cells, CD4 or CD8 T cells and various changes in CD4: CD8 ratio after treatment. These results indicate that the effect of treatment on maintaining homeostasis of T cell subsets was negligible, despite substantial changes in total T cell numbers after treatment. All values were compared with control values.

Figure 28: Ratio analysis of B cells and myeloid cells (NK, NKT and macrophages) in peripheral blood of human patients undergoing LHRH-agonist treatment demonstrated varying degrees of change in the subset. NK, NKT and macrophage ratios were relatively constant after treatment, but the percentage of B cells decreased in 4 of 9 patients.

29: Total cell number analysis of B cells and myeloid cells in peripheral blood of human patients after treatment showed NK (5 of 9 patients), NKT (4 of 9 patients) and macrophages (9) after treatment 3 of the patients) clearly showed an increase in the number of cells. The B cell number did not show a clear trend: levels were increased in 2 of 9 patients, unchanged in 4 of 9 patients, and decreased in 3 of 9 patients. .

30A and B: The major change seen after LHRH-agonist treatment was in the T cell population of peripheral blood. In particular, the ratio of naive (CD45RA ⁺ ) CD4 ⁺ cells was selectively increased, and the ratio of naïve cells (CD45RA ⁺ ) to memory cells (CD45RO ⁺ ) in the CD4 ⁺ T cell subset increased in 6 of 9 human patients. It was.

31: Reduction of impedance of skin with various laser pulse energies. When plotting and writing data, the skin impedance was reduced in the skin stimulated with energy as low as 10 mJ.

32: Penetration of medicines through the skin. Insulin was used as a sample drug, and the laser irradiation significantly increased the permeability of the skin.

33: Change in fluorescence of skin over time after addition of 5-aminolevulinic acid (ALA) and one impulse transient to skin. A strong peak appeared at about 640 nm and was highest after 210 minutes (dashed line) of treatment.

34: Change in fluorescence of skin over time after addition of 5-aminolevulinic acid (ALA) without impulse transition. Intensity changed little at different time points.

35: Comparison of fluorescence changes in skin after addition of 5-aminolevulinic acid (ALA) and one impulse transition under various peak stresses. The permeability of the stratum corneum depends on the peak stress.

The present disclosure relates to methods for improving the vaccine response of a patient. This is achieved by qualitatively and quantitatively restoring the peripheral T cell population, especially at the intrinsic T cell level. Intrinsic T cells are those that have not yet contacted an antigen and therefore have a wide range of basal specificities, ie, can respond to any one of a wide variety of antigens. As a result of the procedure of the present invention a large population of native T cells is able to respond to the antigen administered in the vaccine.

In a preferred embodiment, blocking of signaling to the sex steroid mediated thymus produces this population of native T cells by reactivation of the atrophic thymus. This destruction reverses the recipient's hormonal status. A preferred method of causing destruction is through castration. Castration methods include, but are not limited to, chemical castration and surgical castration.

A preferred method of thymic reactivation is to block the stimulatory effects of LHRH on the pituitary gland, resulting in the loss of gonadotropin FSH and LH. These gonadotropins generally act on the gonads to release sex hormones, especially estrogens in women and testosterone in men; This release is blocked by the loss of FSH and LH. A direct consequence of this is an immediate decrease in the plasma levels of sex steroids, with the result that the suppression signal to the thymus is gradually released. The degree and pattern of thymic re-development can be enhanced by infusion of CD34 ⁺ hematopoietic cells (ideally from autologous or genetic syngeneic tissue).

In the present invention, any animal species (including humans) mature by the induction of sex steroids and possess the immune system can be used, for example mammals and marsupials, preferably large mammals, most preferably humans.

The terms "regeneration", "reactivation" and "reconstitution" and analogues thereof are interchangeable herein and mean that the shrunken thymus is restored to its active state.

As used herein, "castration" means a significant reduction or elimination of the production and distribution of steroids in the body. This effectively returns the patient to the pre-pubertal state when the thymus is fully functional. Surgery castration removes the patient's gonads.

A less permanent form of castration is by a method of administering a chemical over a period of time, referred to herein as "chemical castration". There are a variety of chemicals that can function in this way. During and after the chemical delivery, the patient's hormone production is stopped. Preferably castration is returned at the end of chemical delivery.

The present invention is useful for many diseases. For example, over time, the immune system will essentially run out of native cells. As the number of cells decreases, the number of intrinsic cells capable of generating a response is insufficient, so the response to the antigen present in the vaccine is also reduced. Reactivation of the thymus produces a new, large population of native T cells that can respond appropriately to vaccines.

In addition, patients with a disease or condition, such as a cancer that elicits an prolonged immune response, may have antigen-specific "clonal burnout". While the patient's native T cells initially respond appropriately to foreign antigens on the tumor, thereby producing various clones of the cells to continue making antibodies with foreign antigen specificity, and after a long time these clones will lose production capacity. In patients with atrophic thymus, the population of native T cells capable of continuing the immune response tends to be greatly reduced and rarely exists, and the immune response is essentially extinct or unable to remove the tumor from the patient. Function at the level Reactivation of the thymus can prevent or alleviate clonal exhaustion by creating a large population of native T cells that can respond to foreign tumor antigens.

Destruction of signaling into the thymus mediated by sex steroids

As will be readily appreciated, signaling to the thymus mediated by sex steroids can be disrupted within the scope of methods known to those skilled in the art, some of which are described herein. For example, inhibition of sex steroid production or blocking of one or more sex steroid receptors in the thymus may be a desired signal by administration of a sex steroid agonist or antagonist, or by an active (antigen) or passive (antibody) anti-steroidal steroid vaccine. Will destroy the transmission. Inhibition of sex steroid production can also be achieved by administration of one or more sex steroid analogs. In some clinical situations, permanent removal of the gonads via physical castration may be appropriate.

In a preferred embodiment, signaling to the thymus mediated by sex steroids is disrupted by administration of sex steroid homologues, preferably homologues of luteinizing hormone secretory hormone (LHRH). Sex steroid homologs and their use in therapy and chemical castration are well known. Such homologues include, but are not limited to, agonists of the LHRH receptor (LHRH-R), such as: Buserelin (from Hoechst), Cystorelin (from Hoechst) , Decapeptyl (registered trademark: Debiopharm; manufactured by Ipsen / Beaufour), Deslorelin (from Balance Pharmaceuticals), Gonadorelin (Ayer Ayerst), Goserelin (registered trademark: Zoladex; Zeneca), Histrelin (Ortho), Leuprolide (registered trademark: Lupron ; Abbott / TAP), Leuprorelin [Plosker et al.], Lutrelin (Wyeth), Miterelin [W0 9118016], Nafarel Nafarelin (from Syntex) and Triptorelin (US Pat. No. 4,010,125). In addition, LHRH homologues include, but are not limited to, the following antagonists of LHRH-R: Abarelix® from Plenaxis® from Pracis and Cetrorelix (Registered trademark: Zentaris). Also included are combinations of agonists, combinations of antagonists, and combinations of agonists with antagonists. The disclosures of each of the aforementioned documents are incorporated herein by reference. It is presently preferred that the homologue is Deslorelin (described in US Pat. No. 4,218,439). For a more detailed list, see Vickery et al., 1984.

In a preferred embodiment, the LHRH-R antagonist is delivered to the patient, followed by the LHRH-R agonist. This protocol inhibits or limits any sharp rise in sex steroid production that may be produced by administration of agonists prior to a decrease in sex steroid production. In other embodiments, LHRH-R agonists that use little or no sudden increase in sex steroid production are used, with or without prior administration of LHRH-R antagonists.

Stimulation for thymus reactivation is based primarily on suppressing the effects of sex steroids and / or the direct effects of LHRH homologues, but it is useful to include additional substances that can act to alleviate the thymus effect in concert. can do. Such compounds include interleukin 2 (IL2), interleukin 7 (IL7), interleukin 15 (IL15), members of the epithelial and fibroblast growth factor family, hepatocellular factor, granulocyte colony stimulating factor (GCSF) and keratinocyte growth factor (KGF). ), But is not limited thereto. It is contemplated that such additional compound (s) will be provided only once upon initial LHRH homolog use. However, any one or combination of these agents may be further administered at any time to further stimulate the thymus. In addition, steroid receptor based modulators that can be specifically targeted thymus can be developed and used.

Pharmaceutical composition

The compounds used in the present invention can be supplied with or without any pharmaceutically acceptable carrier. Examples of carriers include physiologically compatible coatings, solvents and diluents. For parenteral, subcutaneous, intravenous and intramuscular administration, the composition can be protected, for example, by encapsulation. Alternatively, the composition may be supplied with a carrier that protects the active ingredient (s) while allowing slow release of the active ingredients. Many polymers and copolymers are known in the art, such as various versions of lactic / glycolic acid copolymers, used to prepare sustained release formulations. See, for example, US Pat. No. 5,410,016 which uses modified polymers of polyethylene glycol (PEG) as biodegradable coatings.

Formulations to be orally delivered can be prepared as liquids, capsules, tablets and the like. These compositions may include, for example, excipients, diluents and / or coatings that protect the active ingredient (s) from degradation. Such agents are well known.

In certain formulations, other compounds may also be included that do not negatively affect the activity of the LHRH homologue. Examples of such compounds are the various growth factors and other cytokines described herein.

Dosage

LHRH homologues can be administered in a single dose that will last for a period of time. Preferably, the formulation will be effective for 1 to 2 months. Standard dosages vary depending on the type of homologue used. Generally, the dosage is about 0.01 pg / kg to about 10 mg / kg, preferably about 0.01 mg / kg to about 5 mg / kg. Dosage depends on the LHRH homologue or vaccine used. In a preferred embodiment, the dosage is made to last for as long as the periodic epidemic lasts. For example, a "cold season" typically occurs in winter. Formulations of LHRH homologues can be made and delivered as described herein to protect the patient for at least two months starting at the beginning of the cold season and delivered every two months or more until the risk of infection is reduced or disappeared.

The formulations may be prepared to enhance the immune system. Alternatively, the formulation may be prepared to specifically prevent infection by cold virus while enhancing the immune system. The latter formulation will include GM cells that have been adapted to develop resistance to cold virus (see below). GM cells can be administered spatially and / or temporally with the LHRH homologue preparation or separately. As with non-GM cells, multiple doses may be administered to a patient over a period of time to protect against and prevent infection with the cold virus over the cold season period.

Delivery of substances for chemical castration

Delivery of the compounds of the present invention can be accomplished through a number of methods known to those skilled in the art. One standard method of administering a chemical inhibitor that inhibits signaling to the thymus mediated by sex steroids is to use a single dose of LHRH agonist that is effective for three months. In this case, a simple intravenous or intramuscular injection will not be sufficient because the agonist can be cleared from the patient's body before three months have passed. Instead, depot injections or implants may be used, or other inhibitor delivery means that will allow sustained release of the inhibitor. Likewise, methods for increasing the half-life of the inhibitor in the body, such as chemical modification, can be used while maintaining the functions required herein.

Examples of even more useful delivery mechanisms include, but are not limited to, laser irradiation of the skin, and high pressure impulse transients (also called stress waves or impulse transients) on the skin, each of the methods being with or without a carrier at the same locus. The compound (s) is accompanied by the placement of the compound (s), or after each of the above methods, with or without the carrier at the same locus. A preferred method of this placement is a patch that is placed and kept on the skin for the duration of treatment.

One delivery means utilizes a laser beam that is specifically focused and laser irradiation at an appropriate wavelength to produce small punctures or alterations in the skin of the patient. See U.S. Patent 4,775,361, U.S. Patent 5,643,252, U.S. Patent 5,839,446, and U.S. Patent 6,056,738, which are incorporated herein by reference in their entirety. In a preferred embodiment, the laser beam has a wavelength of 0.2 to 10 microns. More preferably, the wavelength is about 1.5 to 3.0 microns. Most preferably, the wavelength is about 2.94 microns. In one embodiment, the laser beam is focused by a lens that forms an irradiation spot on the skin through the epidermis of the skin. In a further embodiment, the laser beam is focused to form an irradiation spot only through the stratum corneum of the skin.

As described herein, "ablation" and "perforation" refer to pores in the skin. These pores can vary in depth, for example, the pores can only penetrate the stratum corneum, always penetrate into the capillary layer of the skin, or end the penetration at some point between the stratum corneum and capillary layer. As used herein, “altered” means a change in skin structure that increases the permeability of the skin without the creation of pores. Like the perforation, the skin can be changed to any depth.

Several factors can be considered to define the laser beam, including wavelength, energy fluence, pulse temporal width, and irradiation spot-size. In a preferred embodiment, the energy fluence is between 0.03 and 100,000 J / cm ² . More preferably, the energy fluence is between 0.03 and 9.6 J / cm ² . The light wavelength depends in part on the laser material such as Er: YAG. The pulse duration is the result of the pulse width formed by, for example, lined capacitors, flashlamps and laser rod materials. This pulse width is optionally between 1 fs (femtoseconds) and 1,000 μs.

According to the method of the invention, the perforations or alterations formed by the laser need not be produced by a single pulse from the laser. In a preferred embodiment, puncture or alteration through the stratum corneum is generated using multiple laser pulses, each of which punctures or modifies only a portion of the target tissue thickness.

As a result, one skilled in the art can determine the energy of a single pulse and divide it by the desired number of pulses to approximate the energy needed to puncture or alter the stratum corneum in multiple pulses. For example, if a spot of a particular size requires 1 J of energy to produce a puncture or alteration through the entire stratum corneum, one of skill in the art would know 10 pulses, each having 1/10 J of energy. Can be used to make quantitatively similar perforations or deformations. In a preferred embodiment the pulse repetition rate coming from the laser is preferred because the patient does not move the target tissue during the laser irradiation period (human response time is 100 ms in size) and the heat generated during each pulse is not significantly spread. Must be such that a complete puncture is produced in less than 100 ms. Alternatively, the orientation of the target tissue and laser can be mechanically fixed such that a change in target position does not occur for a longer irradiation time.

To perforate the skin in a manner that sheds little or no blood, the skin can be perforated or altered through an outer surface, such as the stratum corneum, not as deep as the capillary layer. The laser beam is precisely focused on the skin such that the beam diameter at the skin is in the range of approximately 0.5 microns to 5.0 cm. Optionally, the spot may be slit-shaped with a width of about 0.05 to 0.5 mm and a length of 2.5 mm or less. The width can be of any size and is controlled by the anatomy of the area to be irradiated and the desired rate of puncture of the fluid to be removed or the medicament used. The focal length of the focal lens can be any length, but in one embodiment this focal length is 30 mm.

By varying the wavelength, pulse length, energy fluence (which is a function of laser energy output (in J)) and the size of the light beam at the focus point (cm ² ), and the irradiation spot size, ablation strain (perforation) and non-ablation The effect on the stratum corneum between deformations (changes) can be varied. Both excision and non-ablation alteration of the stratum corneum increases the penetration of the drug later applied.

For example, one can change between ablation tissue-effects and non-ablation tissue-effects by reducing the pulse energy while keeping other variables constant. Using an Er: YAG laser with a pulse length of about 300 μs using a single pulse or radiant energy and irradiating a 2 mm spot on the skin, a pulse energy of approximately 100 mJ causes partial or complete ablation to the stratum corneum. Any pulse energy of less than approximately 100 mJ causes partial or non-ablation changes to the stratum corneum. Optionally, with multiple pulses, the threshold pulse energy required to elevate the infiltration of body fluids or drug delivery is reduced by a factor approximately equal to the number of pulses.

Alternatively, by reducing the spot size while keeping other variables constant, one can change between ablation tissue-effect and non-ablation tissue-effect. For example, dividing the spot area in half will divide the energy needed to achieve the same effect. For example, the radiant output of the laser can be irradiated to 0.5 micron by coupling into a microscope (eg, available from Nikon, Inc., Melville, NY). In this case, the light beam can be focused to the spot at the size of the microscope's resolution limit, which can be about 0.5 microns in size. In fact, when the ray profile is normal, the directed irradiated area may be less than the measured ray size and may exceed the image resolution of the microscope. In this case it is suitable to use an energy fluence of 3.2 J / cm ² in order to non-abdominally alter tissue, where a spot size of 0.5 micron size would require a pulse energy of about 5 nJ. This low pulse energy is readily available from diode lasers and may be obtained from Er: YAG by, for example, weakening the light beam with an absorption filter such as glass.

Optionally, by varying the wavelength of radiant energy while keeping other variables constant, one can change between ablation tissue-effects and non-ablation tissue-effects. For example, instead of Er: YAG (erbium: YAG; 2.94 micron) lasers, use Ho: YAG (holmium: YAG; 2.127 microns) to reduce the absorption of energy by the tissue so that perforation or alteration occurs less. do.

Picosecond and femtosecond pulses generated by the laser may be used to cause alteration or ablation in the skin. This can be achieved with a tuned diode or associated microchip laser delivering a single pulse in a time span ranging from 1 femtosecond to 1 ms (incorporated herein by reference in its entirety, which discloses the use of pulse lengths up to 1 femtosecond). See D. Stern et al., “Corneal Ablation by Nanosecond, Picosecond, and Femtosecond Lasers at 532 and 625 nm,” Corneal Laser Ablation, Vol. 107, pp. 587-592, 1989).

Another method of delivery results in the use of high pressure impulse transients on the skin to make them permeable. See US Pat. No. 5,614,502 and US Pat. No. 5,658,892, which are incorporated herein by reference in their entirety. High pressure impulse transients, eg, stress waves with specific rise times and peak stresses (or pressures) (eg, laser stress waves generated by a laser (LSW)) are disclosed herein through epithelial tissue layers such as the stratum corneum and mucosa. The transport of compounds such as these can be achieved stably and efficiently. These methods can be used to deliver compounds of a wide range of sizes regardless of their total charge. In addition, the impulse transients used in the present invention avoid tissue damage.

Prior to exposure to an impulse transient, the epithelial tissue layer, eg, the stratum corneum, will be impermeable to the foreign compound, which inhibits the compound from dispersing into cells lying underneath the epithelial layer. The epithelial layer can be exposed to an impulse transient to allow the compound to diffuse through the epithelial layer. In general, the rate of diffusion is expressed by the nature of the impulsive transient and the size of the compound being delivered.

The rate of penetration through specific epithelial tissue layers, such as the stratum corneum of the skin, can also be influenced by several other factors, including pH, metabolism of the skin stromal tissue, pressure differences between areas outside the stratum corneum and areas within the stratum corneum, as well as anatomical sites and physical conditions of the skin. Depends on. The physical conditions of the skin also depend on health, age, sex, race, skin care and medical history. For example, prior contact with organic solvents or surfactants affects the physical conditions of the skin.

The amount of compound delivered through the epithelial tissue layer depends on the length of time the epithelial layer remains permeable and the size of the surface area of the epithelial layer that has become permeable.

The characteristics and characteristics of the impulse transients are controlled by the energy source used to produce them. See WO 98/23325, incorporated herein by reference. However, its properties are modified by the linear and nonlinear properties of the coupling medium in which they propagate. Linear attenuation caused by the coupling medium predominantly attenuates the high frequency components of the impulse transient. This correspondingly increases the rise time of the impulse transient while reducing the bandwidth. On the other hand, the nonlinear nature of the coupling medium reduces the rise time. The decrease in rise time is a result of the speed of sound and particle velocity against stress (pressure). As the stress increases, sonic velocity and particle velocity also increase. This makes the leading edge of the impulse transition more steep. The relative strength, nonlinear coefficients, and peak stress of the linear attenuation determine how long the wave must propagate to substantially increase the rise time.

The rise time, size and duration of the impulse transients are chosen to produce a non-destructive (ie non-shock wave) impulse transient that temporarily increases the permeability of the epithelial tissue layer. In general, the rise time is at least 1 ns, more preferably about 10 ns.

The peak stress or pressure of the impulse transients depends on the different epithelial tissues or cell layers. For example, in order to transport the compound through the stratum corneum, the peak stress or pressure of the impulse transition should be set to at least 400 bar, more preferably at least 1,000 bar and less than about 2,000 bar.

For epithelial mucosa, the peak pressure should be set at 300 bar to 800 bar, preferably 300 bar to 600 bar.

Impulsive transients preferably have a duration of tens of ns and thus interact with epithelial tissue for a very short time.

After interaction with the impulse transient, epithelial tissue is not permanently damaged and remains permeable for about 3 minutes.

In addition, this method involves only applying a slightly differentiated high amplitude pulse to the patient. The number of impulse transients administered to a patient is typically less than 100, more preferably less than 50 and most preferably less than 10. When using multiple optical pulses to create an impulse transient, the duration between successive pulses is 10 to 120 seconds, which is long enough to prevent permanent damage of epithelial tissue.

The characteristics of the impulse transient can be measured by standard methods of the prior art. For example, peak stress or pressure, and rise time can be found in Doukas et al., Ultrasound Med. Biol., 21: 961 (1995)] can be measured using the polyvinylidene fluoride (PVDF) converter method.

Impulse transients are produced by various energy sources. The physical phenomena involved in the initiation of the impulse transient are generally selected from three different mechanisms: (1) thermoelastic development, (2) optical breakdown, or (3) ablation.

For example, an impulse transition can be initiated by applying a high energy source to ablate a target material, which is then coupled with an epithelial tissue or cell layer by a coupling medium. The coupling medium can be, for example, liquid or gel as long as it is nonlinear. Thus, water, oils such as castor oil, isotonic mediums such as phosphate buffered saline (PBS), or gels such as collagen gels can be used as coupling medium.

In addition, the coupling medium may comprise a surfactant that improves transport by, for example, prolonging the time that the stratum corneum remains permeable to the compound after generation of the impulse transition. Surfactants can be, for example, ionic detergents or nonionic detergents, and therefore can include, for example, sodium lauryl sulfate, cetyl trimethyl ammonium bromide and lauryl dimethyl amine oxide.

The absorption target material acts as a photoinduced transducer. After absorbing light, the target material rapidly thermally expands or ablates to initiate the impulse transition. Typically, metal and polymer films have high absorption coefficients in the visible and ultraviolet spectral regions.

Various types of materials can be used as target materials with the laser beam as long as they absorb light sufficiently at the laser wavelength used. The target material may consist of a metal such as aluminum or copper, polystyrene, for example a plastic, ceramic, or highly concentrated dye solution such as black polystyrene. The target material should have a dimension larger than the cross sectional area of the applied laser energy. In addition, the target material should be thicker than the light transmission depth so that light does not hit the skin surface at all. The target material must also be thick enough to provide mechanical support. The target material may also be made of metal and typical thicknesses will be 1/32 to 1/16 inch. For plastic target materials, the thickness will be 1/16 to 1/8 inch.

Impulse transients can also be improved by using confined ablaition. In limiting ablation, a laser beam transparent material, such as a quartz optical window, is located proximate to the target material. Restriction of the plasma generated by ablation of the target material with a transparent material increases the coupling coefficient with size (see Fabro et al., J. Appl. Phys., 68: 775,1990). The transparent material can be quartz, glass or transparent plastic.

Since the gap between the target material and the limiting transparent material can expand the plasma to reduce the momentum imparted to the target, it is desirable to initially combine the target material with the transparent material using a liquid adhesive such as carbon-containing epoxy. Can prevent gaps.

The laser beam can be generated by standard light modulation techniques known in the art, for example using a Q-switched or mode-locked laser by an electro- or acoustic-optical device. Can be. Commercial standard lasers that can operate in pulsed mode in the infrared, visible and / or infrared spectrum include Nd: YAG, Nd: YLF, C0 ₂ , excimers, dyes, Ti: sapphire, diodes, holmium (and others). Earth materials), and metal-vapor lasers. The pulse width of these light sources is adjustable and can vary from tens of picoseconds (ps) to hundreds of microseconds. When used in the present invention, the light pulse width can vary from 100 ps to about 200 ns, preferably from about 500 ps to 40 ns.

Impulse transients can also be produced by in vitro crushers (an example is described in Coleman et al., Ultrasound Med. Biol., 15: 213-227,1989). This impulse transient has a rise time of 30 to 450 ns, which is longer compared to the impulse transient generated by the laser. In order to form an impulse transient with a rise time appropriate for the new method using an in vitro crusher, the impulse transient is propagated in a nonlinear coupling medium (eg, water) at the distance determined by Equation 1 above. For example, using a crusher that produces an impulse transient with a rise time of 100 ns and a peak pressure of 500 bar, the distance that the impulse transient must pass through the coupling medium before contacting the epithelial cell layer is approximately 5 mm.

A further advantage of this approach to shaping the impulse transients produced by the crusher is that the wave's stretch component will widen or dampen as a result of propagation through the nonlinear coupling medium. This propagation distance should be adjusted such that the pressure of the stretch produces an impulse transient that is only about 5-10% of the peak pressure of the compressive component of the wave. Thus, a standardized impulse transient will not damage tissue.

The type of crusher used is not important. Electrohydraulic, electromagnetic or piezoelectric crushers can be used.

Impulse transients can also be produced using transducers, such as piezoelectric transducers. Preferably, the transducer is in direct contact with the coupling medium and rapidly converted after application of an optical, thermal or electric field to produce an impulse transient. For example, dielectric breakdown may be used, which is typically a high voltage spark or piezoelectric transducer (similar to that used in certain in vitro crushers, see Coleman et al., Ultrasound Med. Biol., 15: 213-227 , 1989). In the case of piezoelectric transducers, the transducers expand rapidly after application of an electric field, causing a rapid transition in the coupling medium.

In addition, impulse transients may also be generated by optical fibers. Fiber optic delivery systems are particularly adjustable and can be used to generate impulsive transients where it is difficult to reach by irradiating target material located adjacent to the epithelial tissue layer. When this type of delivery system is optically coupled with the laser, it is preferred that it can be integrated with the catheter and associated flexible device and used to irradiate most organs of the human body. In addition, in order to initiate an impulse transient with the desired rise time and peak stress, the wavelength of the light source can be easily adjusted to be appropriately absorbed in the particular target material.

Alternatively, the energetic material may generate an impulse transient in response to the impulse provided. The provider can provide an energetic material by causing an electrical discharge or spark.

Hydrostatic pressure can be used with an impulse transient to improve the transport of the compound through the epithelial tissue layer. Since the effect induced by the impulse transient lasts for several minutes, the rate of transport of the drug, which is passively diffused through the epithelial cell layer according to the concentration gradient, applies hydrostatic pressure to the surface of the epithelial tissue layer, for example, the stratum corneum of the skin after applying the impulse transient. Can be increased by applying.

Improving Inoculation Response

With the methods disclosed herein, the sex steroid induced atrophic thymus has been remarkably restored to approximately its optimal prepubertal capacity in all terms that are presently structurally and functionally defined. This includes the number, type and ratio of all T cell subpopulations. Also included are complex interstitial cells and their three-dimensional structures that make up the thymus microenvironment required to produce T cells. Newly generated T cells migrate from the thymus and restore peripheral T cell concentration and function.

Reactivation of the thymus can be aided by the addition of CD34 ⁺ hematopoietic stem cells (HSC) and / or epithelial hepatocytes before the thymus initiates restoration. Ideally these cells are self-derived or genetic syngeneic and obtained from patients or twins prior to thymic reactivation. HSCs can be obtained by sorting CD34 ⁺ cells from the patient's blood and / or bone marrow. The number of HSCs may be, for example, incubated in Stem Cell Growth Factor for cells recovered by administering G-CSF (Neupogen, Amgen) to the patient prior to cell recovery and / or in patients with G-CSF after CD34 ⁺ cell supplementation. It can be improved in various ways, including administering, but is not limited thereto. Alternatively, CD34 ⁺ cells need not be sorted from blood or BM if their subjects are improved by prior injection of G-CSF into the patient.

Within 3-4 weeks of initiation of the signal block mediated by sex steroids (approximately 2-3 weeks after initiation of LHRH treatment), the first new T cells appear in the bloodstream. However, full development of T cell pools can take 3-4 months. Inoculation can, in principle, begin some time after the appearance of newly generated new cells, but 4 to 4 days after initiation of LHRH treatment if sufficient new T cells are produced to show a strong response and any necessary post-thymic maturation is performed. It is desirable to wait up to six weeks before starting the inoculation.

This process may be accompanied by any other form of immune system stimulation such as adjuvant, adjuvant molecules and cytokine treatment. Useful cytokines, for example, include the common immune growth factors interleukin 2 (IL2), IL4 for biasing the response to Th2 (humoral immunity), and Th1 (cell mediated, inflammatory response). Include, but are not limited to. Examples of auxiliary molecules include, but are not limited to, inhibitors of CTLA4 that promote CD28 / B7.1, B7.2 stimulation pathways that are generally inhibited by CTLA4, thereby improving the general immune response.

Small animal research

Materials and methods

animal

CBA / CAH and C57B16 / J male mice were purchased from Central Animal Services (Monash University) and bred under conventional conditions. The ages of the mice ranged from 4-6 weeks to 26 months and are indicated in relevant locations.

castration

0.3 mg of xylazine in salt water (Rompun; Bayer Australia Ltd., Botany, Australia) and ketamine hydrochloride (Ketalar; Parke, Botany, USA) Animals were anesthetized by intraperitoneal injection of 0.3 ml of a 1.5 mg solution from Davis (Parke-Davis). The scrotum was dissected to expose the testicles, tied with sutures, and castration was performed with surgery to remove the surrounding adipose tissue.

Incorporation of Bromodeoxyuridine (BrdU)

BrdU (Sigma Chemical Co., St. Louis, MO) (100 mg / kg body weight in 100 μl PBS) was injected intraperitoneally into mice twice at 4 hour intervals. Control mice were injected with vehicle only. One hour after the second injection, the thymus was dissected to prepare a cell suspension for FACS analysis or immediately Tissue Tek ( OCT ) compound, Miles INC, Indiana, USA Product) and was rapidly frozen in liquid nitrogen and stored at -70 ° C until use.

Flow Cytometric analysis

Mice were killed by choking with C0 ₂ and the thymus, spleen and mesenteric lymph nodes dissected. The organ was gently filtered through a 200 μm sieve in a cold PBS / 1% FCS / 0.02% azide solution, centrifuged (650 g, 5 min, 4 ° C.) and resuspended in PBS / FCS / Az. Spleen cells were washed by incubation at 4 ° C. for 10 min in erythrocyte lysis buffer (8.9 g / L ammonium chloride) and then resuspended in PBS / FCS / Az. Cell concentration and viability were measured in duplicate using a hemocytometer and ethidium bromide / acridin orange and under a fluorescence microscope (Axioskop; Carl Zeiss, Oberkochen, Germany). .

For the three-color immunofluorescence, the anti-thymocyte -αβTCR-FITC or anti -γδTCR-FITC, anti -CD4 ^- as APC (All Zen Farmington (Pharmingen) products based in San Diego, Calif.) ^- PE and anti -CD8 After labeling, flow cytometry was performed. Spleen and lymph node suspensions were labeled with αβTCR-FITC / CD4 ^- PE / CD8 ^- APC or B220-B (from Sigma) including CD4 ^- PE and CD8 ^- APC. B220-B is Carl Tag Laboratories, Inc. in Burlingham, California. It was detected by streptavidin-tri-color conjugate purchased from Caltag Laboratories, Inc.

For BrdU detection, cells were surface labeled with CD4 ^- PE and CD8 ^- APC, then fixed and infiltrated as described previously (Carayon and Bord, 1989). In summary, the stained cells were fixed overnight in 1% PFA / 0.01% Tween-20 at 4 ° C. The washed cells were incubated at 37 ° C. for 30 minutes in 500 μl of DNase (100 Kunitz unit, Boehringer Mannheim, West Germany) to denature DNA. Finally, cells were incubated with anti-BrdU-FITC (Becton-Dickinson).

For four-color immunofluorescence, thymic cells were labeled for CD3, CD4, CD8, B220 and Mac-1, and the whole was detected with anti-rat Ig-Cy5 (Amersham, UK) and TN (negative) cells) were gated for analysis. As described above [Godfrey and Zlontnik, 1993], streptavidin-tri-collar (Cal., USA) after staining cells with CD25-PE (from Farmingen) and CD44-B (from Farmingen) Tag product). Subsequently, BrdU detection was performed as described above.

Samples were analyzed on FacsCalibur (Becton-Dickinson). According to 0 ° and 90 ° light scattering profiles, viable lymphocytes were gated and data analyzed using Cell quest software (Becton-Dickinson).

Immunohistochemistry

Frozen thymic sections (4 μm) were cut in 100% acetone immediately after cutting using a frozen microcutter (Leica).

For two-color immunofluorescence, MTS6, 10, 12, 15, 16, 20, 24, 32, 33, 35 and 44 monoclonal antibody panels produced by the laboratory [Godfrey et al., 1990; Table 1] was used to double-label the fragments and to evaluate the co-expression of epithelial cell determinants using a multivalent rabbit anti-cytokeratin antibody (Dako, Calif., Calif.). Bound mAb was revealed by both FITC-conjugated anti-rat Ig (manufactured by Silenus Laboratories), and the anti-cytokeratin was found to be TRITC-conjugated goat anti-rabbit Ig (Sirenus labrover) Leeds product).

For BrdU detection, sections were stained with anti-cytokeratin and then stained with anti-rabbit-TRITC or specific mAbs and then identified as anti-rat Ig-Cγ3 (made by Amulsham). Subsequently, BrdU detection was performed as described above [Penit et al., 1996]. In summary, sections were fixed in 70% ethanol for 30 minutes. Semi-dried sections were incubated in 4 M HCl, neutralized by washing with borate buffer (Sigma) and then washed twice in PBS. BrdU was detected using anti BrdU-FITC (Becton-Dickinson).

For tricolor immunofluorescence, sections were labeled for specific MTS mAb and anti-cytokeratin. Then BrdU detection was performed as described previously.

Sections were analyzed using Leica phosphor and a Nikon confocal microscope.

Moving research

Intraperitoneally in a brine, 0.3 ml of a solution of 0.3 mg of xylazine (Lampen; Bayer Australia Limited, Botney N. Double Oil) and 1.5 mg of ketamine hydrochloride (Ketala: Parke-Davis, Botney N. Double Oil, Australia) Animals were anesthetized by my injection.

Details on the FITC labeling technique of thymic cells were similar to those described in other documents (Scollay et al., 1980, Berzins et al., 1998). In summary, the thymus lobe was exposed and each lobe was injected with about 10 μl of 350 μg / ml FITC (in PBS). The wound was closed with staples used for surgery and the mice were allowed to warm until complete waking from anesthesia. Mice were killed by choking with C0 ₂ 24 hours after injection and lymphoid organs were excised and analyzed.

After counting the cells the samples were stained with anti ^- CD4 ^- PE and anti-CD8 ^- APC and analyzed by flow cytometry. Migrating cells were identified as live-gated FITC ⁺ cells (missing autofluorescent cells and doublets) expressing CD4 or CD8. Percentages of FITC ⁺ CD4 and CD8 cells were totaled for lymph nodes and spleen, respectively. Add to provide percentage transfer. Calculations for the daily release rate are reported in Berzins et al. (1998).

Assay results for experiments performed with at least three experiments using data analyzed using the unpaired student't 'test or the nonparametrical Mann Whitney test Statistical significance between was determined. Experimental values significantly different from the control are shown as follows: * p ≦ 0.05, ** p ≦ 0.01 and *** p ≦ 0.001.

result

Age influence on thymic cell population

(i) Thymus Weight and Thymus Cell Number

As age increased, there was a very significant (p ≦ 0.0001) decrease in both thymus weight (FIG. 1A) and total thymic cells (FIG. 1B). The relative weight of the thymus (mg thymus / g body weight) in young mature mice has an average value of 3.34, decreasing to 0.66 after 18 to 24 months of age (due to fat accumulation, the exact calculations are limited). The decrease in thymus weight can be attributed to the decrease in the total number of thymic cells: thymus from one to two months old is 6.7 × 10⁷dog Contains thymic cells, ~ 4.5 x 10 at 24 months of age⁶Decreases in canine cells. Regeneration occurs when castration eliminates the effects of sex steroids on the thymus, and the thymus after 4 weeks of castration is equivalent to the level of young mature mice in both weight and cell fidelity (FIGS. 1A and 1B). Interestingly, after two weeks of castration, the number of thymic cells increased significantly (p ≤0.001) and increased (~ 1.2 × 10).⁸), It recovers to normal young mature mouse level after 4 weeks of castration (FIG. 1B).

The decrease in the number of T cells produced by the thymus is not reflected at the beginning and spleen cell numbers remain constant regardless of age (FIG. 2A). Peripheral homeostasis mechanisms are evident because the ratio of B cells to T cells in the spleen and lymph nodes is not affected by the subsequent decrease in age and number of T cells reaching the periphery (FIG. 2B). However, the ratio of CD4 ⁺ to CD8 ⁺ T cells decreased significantly (p ≦ 0.001) with age from 2: 1 at 2 months of age to 1: 1 at 2 years of age (FIG. 2C). After a subsequent increase in the number of T cells reaching castration and distal, no change was observed in the peripheral T cell counts: the ratio of B: T cells in both spleen T cells and both spleen and lymph nodes did not change after castration. (Figures 2A and B). The ratio of reduced CD4 to CD8 at the periphery with age is still apparent after 2 weeks of castration, but completely reverses after 4 weeks of castration (FIG. 2C).

(ii) αβTCR, γδ TCR, CD4 and CD8 expression

In order to determine whether the decrease in the number of thymic cells with increasing age is the result of specific cell population depletion, thymic cells were labeled with characteristic markers and analyzed for a separate subpopulation. This also enabled a kinetic analysis of thymic regrowth after castration. The percentage of major thymic cell subpopulations was compared to that in normal young mature mouse thymus (FIG. 3) and found to remain constant regardless of age. In addition, further segmentation of thymic cells by expression of αβTCR and γδTCR revealed no change in the proportion of these populations with increasing age (data not shown). After 2 and 4 weeks of castration, the thymic cell subpopulation remained at the same rate, and the number of thymic cells increased up to 100-fold after castration, indicating a simultaneous increase in subsets of all thymic cells rather than progression in development. .

Thus, a decrease in the number of cells present in the thymus of older animals is believed to be the result of a balanced decrease in the phenotype of all cells, with no significant changes detected in the T cell population. Thymic regeneration occurs in a simultaneous manner and replenishes all T cell subpopulations simultaneously rather than sequentially.

Thymic Cell Proliferation

As shown in FIG. 4, 15 to 20% of thymic cells proliferate in 4 to 6 week old mice. Most of them (˜80%) are DPs with TN subsets making up the second largest population of ˜6% (FIG. 5A). Thus, most of the fractions appear in subcapsules and cortex by immunohistochemistry (data not shown). Some fractions appearing in the medulla region by FACS analysis show the proportion of dividing SP cells (9% of CD4 T cells and 25% of CD8 T cells) (FIG. 5B).

Although the number of cells in the thymus of older mice is significantly reduced, the proliferation of thymic cells remains constant and decreases to 12-15% at 2 years of age (Figure 4), and the phenotype of the proliferating population is similar to that of the 2-month old thymus. (FIG. 5A). Immunohistology revealed a one year old fraction and reflects the fraction seen in young mature mice; However, proliferation at 2 years of age occurs mainly in the outer cortex and surrounding blood vessels (data not shown). After two weeks of castration, the number of thymic cells increased significantly, but there was no change in the percentage of proliferating thymic cells, which in turn indicates a simultaneous increase in cells (FIG. 4). Immunohistology revealed localization of thymic cell proliferation and the degree of division of cells in a situation similar to that in the thymus of two months old mice two weeks after castration (data not shown). In analyzing the proportion of each subpopulation representing the proliferating population, there was a significant (p ≦ 0.001) increase in the proportion of CD8 T cells in the proliferating population (1% at 2 months and 2 years old, castration 2 Increase to ˜6% after week) (FIG. 5A).

5B illustrates the extent of proliferation in each subset of young mice, older mice and castrated mice. There is a significant (p ≦ 0.001) decrease in proliferation in the DN subset (35% at 2 months old to 4% at 2 years old). In addition, the proliferation of CD8 ⁺ T cells was significantly reduced (p ≦ 0.001), reflecting the results obtained in immunohistochemistry where fractions in the medulla of older thymus are not apparent (data not shown). The decrease in DN proliferation after 4 weeks of castration does not return to normal young mature mouse levels. However, proliferation in the CD8 ⁺ T cell subsets increased significantly (p ≦ 0.001) after 2 weeks of castration and returned to normal young mature mouse levels after 4 weeks of castration.

Reduction of proliferation within the DN subset was further analyzed using the markers CD44 and CD25. The thymic cell precursor as well as the DN subpopulation contain αβTCR ⁺ CD4 ^- CD8 ⁻ thymic cells, which are downregulated at both co-receptors in metastasis to SP cells [Godfrey & Zlotnik, 1993]. These mature cells by gating the true TN compartment (CD3 ^- CD4 ^- CD8 ^-) was possible to analyze, then depending on the age or castration, there was no difference in growth rate (FIG. 5C). However, analysis of subpopulations expressing CD44 and CD25 showed a significant (p <0.001) decrease in the proliferation of the TN1 subset (CD44 ⁺ CD25 ⁻ ) (in 20% of normal young mature mice aged 18 months). Reduced to about 6%) (FIG. 5D), which recovered after 4 weeks of castration. The decrease in proliferation of the TN1 subset was offset by a significant (p ≦ 0.001) increase in the proliferation of TN2 subpopulations (CD44 ⁺ CD25 ⁺ ) that recovered to normal young mature mouse levels after 2 weeks of castration.

Age influence on thymus microenvironment

Changes in the thymus microenvironment with age were examined by immunofluorescence using an extensive panel of MAbs (MTS series, double labeled with polyclonal anti-cytokeratin Ab).

Antigens recognized by these MAbs were subclassified into three groups: thymic epithelial subsets, vascular-associated antigens and antigens present in both basal and thymic cells.

(i) epithelial cell antigens

Anti-keratin staining (pre-epithelial) of the 2 year old mouse thymus revealed that the general thymus structure was missing, with severe deorganization of epithelial cells and the absence of distinct cortical-medullary junctions. Further analysis with MAb, MTS 10 (water quality) and MTS44 (cortex) showed that there was a substantial decrease in medulla epithelium with the fact that cortical size was significantly reduced with age (data not shown). Regions lacking epithelial cells or keratin negative regions, as evident in anti-cytokeratin markers (KNA's, van Ewijk et al., 1980, Godfrey et al., 1990, Bruijntjes et al., 1993) This was more evident and increased in size in the thymus of older mice. In addition, the appearance of the "cyst-like" structure of the thymus epithelium appeared in the thymus of older mice, especially in the medulla region (data not shown). Fat accumulation, severe reduction in thymus size, and integrative decline in cortical-medullary junctions were crucial in anti-cytokeratin staining (data not shown). The thymus begins to regenerate two weeks after castration. This is evident with thymus lobe size, an increase in cortical epithelium, represented by MTS 44, and localization of the medulla. The medulla epithelium is detected with MTS 10, and after 2 weeks the subbags of epithelium stained with MTS 10 are still dispersed throughout the cortex. After 4 weeks of castration, there is a pronounced cortex and medulla and identifiable cortex-medullary junctions (data not shown).

Markers MTS 20 and 24 are assumed to detect primitive epithelial cells (Godfrey, et al., 1990) and further illustrate the degeneration of older thymus. They are rich in E14 and detect medulla epithelial cell populations isolated in 4-6 week old mice, but increase in intensity even in older thymus (data not shown). After castration, all these antigens were expressed at the same level as the young mature mouse thymus (using MTS 20 and MTS 24, data not shown) and returned to separate sub-bags present at the cortical-medulla junction.

(ii) vascular-associated antigens

The blood-thymus barrier is believed to be responsible for entry of T cell precursors into the thymus and entry of mature T cells out of the thymus and into the periphery. MAb MTS 15 is specific for the endothelium of thymic blood vessels and exhibits a globular diffuse staining pattern (Godfrey, et al., 1990). MTS 15 expression is significantly increased in older thymus, indicating increased vascular frequency and size and prevascular space (data not shown).

The extracellular matrix of the thymus containing important structural and cell adhesion molecules such as collagen, laminin, fibrinogen and the like is detected with MAb MTS 16. The expression characteristics of MTS 16, which are distributed throughout the normal young mouse thymus, become wider and more interconnected in older thymus. Expression of MTS 16 further increased after 2 weeks of castration, but after 4 weeks of castration this expression indicates a situation of 2 months old thymus (data not shown).

(ii) shared antigens

MHC II expression in normal young thymus detected with MAb MTS 6 is very positive (granular) on cortical epithelium and weakly stained in medulla epithelium (Godfrey et al., 1990). Older thymus showed a decrease in MHC II expression, which expression increased substantially after two weeks of castration. After 4 weeks of castration the expression is reduced again and resembles a 2 month old thymus (data not shown).

Thymic cell migration

In young mice about 1% of T cells migrate from the thymus daily (Scollay et al., 1980). We have found that migration occurs even at 2 years of age at the same rate as 14 months old normal young mice (FIG. 5), but the number decreases significantly (p ≦ 0.0001). The ratio of CD4 to CD8 in newly entering thymus increases from ~ 3: 1 in two months to ~ 7: 1 in 26 months old. After 1 week of castration, the number of cells moving to the distal end increased significantly, and the overall rate of migration remained constant at 1-1.5%.

The following examples provide specific views of the method of the present invention, but should not be construed as limiting the invention to the content thereof. For convenience, these examples illustrate the delivery of LHRH agonists to block signaling to sex steroid mediated thymus. However, the scope of the present invention is not limited thereto.

Example 1

Sex steroid resection therapy

Sex steroid resection therapy was given to patients in the form of delivery of LHRH-agonists. It is given in the form of Leucrin (depot injection; 22.5 mg) or Zoladex (Insert; 10.8 mg), which is a single dose effective for 3 months. They are effective enough to reduce sex steroid levels, thus reactivating the thymus. In addition, in some cases it is necessary to deliver inhibitors of adrenal production of sex steroids, such as Cosudex (5 mg / day), one tablet per day during sex steroid resection therapy. Adrenal production of sex steroids accounts for about 10-15% of human steroids.

It took about 1 to 3 weeks to reduce blood steroids to a minimum, and reactivation of the thymus was consistent with this. In some cases, treatment may need to be extended to a second three month infusion / graft.

Example 2

Alternative method of delivery

Alternative methods may be used instead of the three month depot or implant administration of LHRH-agonists. In one example, the patient's skin may be irradiated with a laser, such as an Er: YAG laser, to ablate or alter the skin to reduce the damaging effect of the stratum corneum.

A. Laser Ablation or Alteration: Infrared laser radiation pulses were formed using a solid state pulsed Er: YAG laser consisting of two flat resonator mirrors, Er: YAG crystals as the active medium, a power source and a laser beam focusing means. . The wavelength of the laser beam was 2.94 microns. A single pulse was used.

The operating parameters were as follows: the energy per pulse was 40, 80 or 120 mJ and an energy fluence of 1.27, 2.55 or 3.82 J / cm ² was produced with the beam size at the focus of 2 mm. The pulse duration was 300 ms and produced an energy fluence of 0.42, 0.85 or 1.27 × 10 ⁴ W / cm ² .

Subsequently, any amount of LHRH-agonist was applied to the skin and spread on the irradiation site. The LHRH-agonist may be in ointment form to remain on the irradiation site. Optionally, an occlusive patch is placed on the agonist to keep it on the irradiation site.

A beam splitter is optionally used to split the laser beam and create a plurality of ablation or alteration sites. This allows LHRH-agonists to enter the bloodstream faster through the skin. The number of sites can be predetermined to keep the agonist in the patient's system for about 30 days.

B. Pressure Wave: At the site where the pressure wave will be generated, place an arbitrary amount of LHRH-agonist in a suitable container such as a plastic stretch washer (diameter, about 1 inch; thickness, about 1/16 inch) onto the skin. Put on. The site was covered with a target material such as a black polystyrene sheet about 1 mm thick. A Q-switched solid-state ruby laser (20 ns pulse duration, capable of generating up to 2 J per pulse) was used to generate a laser beam, which caused the laser beam to collide with the target material to produce a single impulse transition. The black polystyrene target completely absorbed the laser radiation, leaving the skin exposed only to the impulse transition and not to the laser radiation. There was no pain from this process. To maintain agonist blood levels, the process can be repeated daily or as many times as required.

Example 3

Selective HSC Administration

In one embodiment, hematopoietic stem cells (HSC) are administered to the patient to accelerate the restoration of the thymus. They may also use HSCs of donor pairs (homologous or heterologous) that are of self-derived or genetic syngeneic but inadequate pairs. In practical cases, the concentration of HSC in the blood of a patient or donor can be improved by collecting cells after 10 μg / kg administration of granulocyte colony stimulating factor (G-CSF) to the patient or donor for 2 to 5 days. CD34 ⁺ cells can be purified from the blood or bone marrow of the patient or donor, preferably using a flow cytometer or immunomagnetic beading. HSCs are identified as CD34 ⁺ in the flow cytometer. Optionally these HSCs are expanded ex vivo using Stem Cell Factor. After about 1 to 3 weeks of LHRH antagonist delivery, at or shortly before the thymus is restored, the patient is optimally infused with HSC at about 2-4 × 10 ⁶ cells / kg. Optionally, G-CSF can be injected into the recipient to help expand HSC.

If HSC is provided from an inappropriate pair of donors, T cell clearance and immunosuppressive treatment can be applied to the recipient to prevent the release of external HSCs. In this example of treatment, the T-antigen is injected for 10 days in a form of daily injection of 15 mg per kg of Atgam (Xeno anti-T cell globulin, manufactured by Pharmacia Upjohn). It can be administered daily in combination of 9 mg / kg tablets after continuous infusion in combination with the cell activation inhibitor cyclosporin A (3 mg / kg). Prevention of T cell reactivation can enhance T cell clearance in combination with a second concentration signal inhibitor such as interleukin or cell adhesion molecule. Such treatment may begin upon or prior to the removal of the sex steroid.

Reactivated thymus takes purified HSCs and converts them into new T cells. When using unpaired donor HSCs, the donor dendritic cells will tolerate any T cells potentially active for the patient by causing deletion by cell necrosis or through tolerance through immune regulatory cells.

Since new T cells are purged with latent autoactive and host active cells positively selected as host thymic epithelium, they can recognize peptides provided by surrounding host APCs and respond to normal infection. Both the patient and the donor's CD34 ⁺ HSCs are expressed in dendritic cells and expressed in the patient's lymphatic organs, increasing the amount of new T cells but ultimately establishing the same immune system as the patient's own immune system.

Example 4

Restoration of the thymus

The activated thymus take HSCs and convert them into new T cells, which migrate into the bloodstream to reconstruct the patient's optimal peripheral T cell reservoir. In particular, a major increase in the concentration and proportion of new T cells occurs, significantly increasing the number of potential reactive cells in the inoculation program. The correct ratio of Th: Tc and Th1: Th2 cells ensures the optimal type of response.

When a new cohort of mature T cells begins to break out of the thymus, blood is taken from the patient to examine the condition of the T cells (and eventually all blood cells). In particular, T cells are examined whether they are Th1 or Th2 and whether they are new or memory cells. They also examine the type of cytokines they produce (Th1 vs Th2).

Immunosuppressive treatment due to the administration of an inappropriate pair of HSCs gradually decreases to allow protection against infection and completes completely in the absence of signs of rejection, which is partly known by the presence of activated T cells in the blood. Since HSCs have strong self-renewal, the hematopoietic chimeras produced would theoretically be stable in situations where an inappropriate pair of HSCs were not used throughout the patient's lifetime.

Example 5

Use of LHRH-agonists to reactivate human thymus

In order to find out that the method of the present invention can reactivate human thymus, the method was used for patients who received chemotherapy for prostate cancer. Patients with prostate cancer were evaluated before and 4 months after sex steroid resection. The results are summarized in Figures 23-27. Collectively, the data indicate that the status of T cells has improved both qualitatively and quantitatively in many patients.

Phenotypic composition of peripheral blood lymphocytes was analyzed in patients undergoing LHRH-agonists for prostate cancer (all over 60 years old) (FIG. 23). Patient samples were analyzed before LHRH-agonist treatment and 4 months after treatment. For all patients the total lymphocyte cell count per ml of blood before treatment was the lowest level of the control. After treatment, 6 of 9 patients showed a substantial increase in the total lymphocyte count (in some cases, a 2-fold increase in the total count was observed). Correspondingly, total T cell numbers increased in 6 of 9 patients. In the CD4 ⁺ subpopulation, this increase was even more pronounced, with 8 of 9 patients showing an increase in CD4 ⁺ T cell levels. Less pronounced trends were observed in the CD8 ⁺ subpopulation, and 4 of 9 patients showed increased levels, although to a lesser extent than CD4 ⁺ T cells.

Effect of LHRH Therapy on T Cell Subpopulation Ratio

Analysis of the patient's blood before and after LHRH-agonist treatment showed no significant change in the overall ratio of T cells, CD4 ⁺ or CD8 ⁺ T cells, and a variable change in the CD4 ⁺ : CD8 ⁺ ratio after treatment (FIG. 24). ). This shows a slight effect of treatment on maintaining homeostasis of T cell subpopulations despite a substantial increase in overall T cell numbers after treatment. All values were compared with those of the control.

Effect of LHRH Therapy on the Proportion of B Cells and Myeloid Cells

Analysis of the proportion of B cells and myeloid cells (NK, NKT and macrophages) in peripheral blood of patients undergoing LHRH-agonist treatment showed different changes in subpopulations (FIG. 25). The proportion of NK, NKT and macrophages remained relatively constant after treatment, while the proportion of B cells decreased in 4 of 9 patients.

Effect of LHRH-agonist therapy on the total number of B cells and myeloid cells

The total cell numbers of B and myeloid cells in peripheral blood after treatment showed clear numbers of NK (5 of 9), NKT (4 of 9) and macrophages (3 of 9) Increased (FIG. 26). B cell numbers showed no obvious trend, 2 of 9 showed increased levels, 4 of 9 did not show any changes, and 3 of 9 showed decreased levels.

Effect of LHRH Therapy on Intrinsic Cell Levels on Memory Cells

Major changes were observed after LHRH-agonist treatment in peripheral blood T cell populations. In particular, the ratio of naive (CD45RA ⁺ ) CD4 ⁺ cells was selectively increased, and the ratio of naive (CD45RA ⁺ ) to memory (CD45RO ⁺ ) in the CD4 ⁺ T cell subpopulation was increased in 6 of 9 patients (FIG. 27). ).

conclusion

Thus, it can be concluded that LHRH-agonist treatment of animals such as humans with atrophic thymus can induce thymus reactivation. It has been observed that the condition of blood T lymphocytes generally improves in patients with prostate cancer who have been given sex steroid resection. It is very difficult to accurately determine whether these cells are only derived from the thymus, but since no other source of mainstream (CD8 αβ chain) T cells has been described, this would be a logical conclusion. Gastrointestinal T cells are mainly TCR γδ or CD8 αα chains.

references

Claims (14)

An inoculation improvement method comprising reactivating the thymus of a patient. The method of claim 1, wherein the thymus of the patient is at least partially inactivated. The method of claim 2, wherein the patient is post puberty. The method of claim 1, further comprising administering hematopoietic stem cells to the patient. The method of claim 4, wherein the hematopoietic stem cells are CD34 ⁺ . The method of claim 4, wherein the hematopoietic stem cells are self-derived or genetic syngeneic. The method of claim 4, wherein the hematopoietic stem cells are allogeneic or heterologous. The method of claim 4, wherein the hematopoietic stem cells are administered at or shortly after the thymus begins to regenerate. 5. The method of claim 4, wherein hematopoietic stem cells are provided at the point at which signaling mediated by sex steroids to the thymus begins to break down. The method of claim 1, wherein the gonad of the patient is removed by surgery to destroy the signaling mediated by sex steroids to the thymus. The method of claim 1, wherein one or more medications are administered to disrupt the signal mediated by the sex steroid to the thymus. The method of claim 11, wherein the medicament is selected from the group consisting of LHRH-agonists, LHRH-antagonists, anti-LHRH vaccines, and combinations thereof. The method according to claim 12, wherein the LHRH-agonist is Eulexin, Goserelin, Leuprolide, Dioxalan derivative, Triptorelin, Meterelin, The method is selected from the group consisting of Buserelin, Histrelin, Nafarelin, Lutrelin, Leuprorelin and Deslorelin. The method of claim 1, which induces an inoculation response by the immune system of the patient corresponding to the response of a patient of prepubertal age.