WO2006043623A1

WO2006043623A1 - Method of preventing or treating hepatopathy

Info

Publication number: WO2006043623A1
Application number: PCT/JP2005/019293
Authority: WO
Inventors: Yoshio Wakatsuki
Original assignee: Kyoto University
Priority date: 2004-10-22
Filing date: 2005-10-20
Publication date: 2006-04-27
Also published as: JP2007320852A

Abstract

It is intended to provide a method of preventing or treating hepatopathy which has an improved convenience and exhibits relatively mild side effects. A method of antigen-nonspecifically preventing or treating mammalian hepatopathy characterized by comprising: (1) the first step of proliferating and activating lymphocytes taken out from a mammal by a specific antigen to thereby prepare lymphocytes which are specific to the antigen; (2) the second step of introducing the antigen-specific lymphocytes obtained in the first step into the liver of the above-described mammal; and (3) the third step of orally administering 1) the above-described antigen per se and/or 2) a substance being equivalent in antigenic activity to the above-described antigen to the above-described mammal and thus stimulating the above-described lymphocytes, which have been introduced in the second step, with the antigen, thereby inducing CD4+FasL+immunomodulatory T cells in the liver tissue.

Description

Methods for preventing or treating liver damage

Technical field

[0001] The present invention relates to a method for preventing or treating liver diseases. More specifically, the present invention relates to a method for preventing or treating liver damage using an immunological anti-inflammatory reaction by orally administering an antigen specific for T cell transfer and transfer τ cell.

Background art

[0002] Hepatitis and the like in mammals, particularly humans, are diseases that can lead to cirrhosis, liver cancer, and death. Liver damage is also caused by an immune response to the self-antigen expressed in the liver as well as hepatitis due to viral infection.

[0003] Today, patients with viral hepatitis and autoimmune liver disease are treated with antiviral agents or immunosuppressive agents such as interferons, reverse transcriptase inhibitors, steroid hormones, etc. . However, these drug treatments are not necessarily treatments based on cell mechanisms that cause liver damage, and side effects that do not have sufficient therapeutic effects cannot be ignored.

[0004] Oral force By administering an antigen, T cells (suppressor T cells, regulatory T cells) that suppress immune inflammatory responses are induced in the body. As an induction mechanism, antigen-specific T cells are removed when a large amount of antigen is administered (clone removal theory). On the other hand, when a small amount of antigen is administered, regulatory T cells are thought to be induced in the intestinal lymph nodes (Peier's patch).

On the other hand, it is known that the liver induces immune tolerance against an antigen that reaches the liver via the portal vein. Also, during transplantation, the liver induces immune tolerance, rejection is less likely to occur, and it is also known as an organ.

[0006] From an anatomical point of view, portal tolerance established by the continuous delivery of antigenic substances derived from food and intestinal microflora to the liver via the portal vein is the tolerance-inducing property of the liver. (Crispe IN, Dao T, Klugewitz Κ, Mehal WZ, Me tz DP. The liver as a site of T— cell apoptosis: graveyard, or killing field? Immunol Re v 2000; 174: 47-62). An example of an application of this characteristic of easily inducing immune tolerance inherent in the liver is, for example, portal spleen transplantation in the treatment of insulin-dependent diabetes (Shapiro, AM et al., N. Engl. Med. 343.230 (2000).

[0007] Recently, various attempts have been made to prevent inflammation by inducing immune tolerance by orally administering to a patient an autoantigen that is thought to cause each disease. For example, rheumatoid arthritis (Trentham, DE et al. Science, 261: 1727 (1993)), multiple sclerosis (Weiner, H 丄. Et al., Science, 2589: 1321 (1993)), type 2 Diabetes (Lalej— Bennis D et al. Efficac y and tolerance of intranasal insulin administered during 4 months in severely hyperg lycaemic Type 2 diabetic patients with oral drug failure: a cross-over study. Diabet Med 2001; 18 (8): 614- 8.), self-administration to patients such as Thurau SR et al. Oral tolerance for treating uv eitis- new hope for an old immunological mechanism.Prog Retin Eye Res 2002; 21 (6): 577-89) Treatment has been performed in which an antigen is administered orally.

[0008] However, these treatment methods require an autoantigen, but it is difficult to obtain and secure a sufficient amount of autoantigen for treatment.

[0009] In addition, because of their various immune tolerance mechanisms (Weiner HL. Oral tolerance, an active immunologic process mediated by multiple mechanisms. J Clin Invest 2000 06 (8): 935-7) Cells with sufficient immunosuppressive effect cannot be induced efficiently.

[0010] In addition, in these methods, in some cases, Bianas E, Heath WR. Oral administration of antigen can lead to the onset of autoimmune disease. Int Rev Immunol 1999 8 (3): 217-28). Disclosure of the invention

Problems to be solved by the invention

[0011] As described above, for patients suffering from severe liver dysfunction due to advanced hepatitis or the like, there is no effective treatment other than liver transplantation. Therefore, development of preventive methods is desired.

Accordingly, an object of the present invention is to solve these problems, and to provide a simpler method for preventing or treating liver damage with relatively few side effects. Means for solving the problem

[0013] In view of the above-mentioned problems of the prior art, the present inventor has found that the above object can be achieved by a method having a specific process capability as a result of extensive research, and has completed the present invention. It was.

That is, the present invention relates to the following method for preventing or treating liver damage.

[0015] 1. A method for non-antigen-specific treatment or prevention of liver damage in mammals, comprising:

(1) a first step of preparing lymphocytes specific to the antigen by proliferating and activating lymphocytes removed from the mammal with a specific antigen;

(2) the second step of introducing the antigen-specific lymphocytes obtained in the first step into the liver of the mammal; and

(3) To the mammal, 1) the antigen itself and Z or 2) the substance having the U antigen activity, such as the antigen, is orally administered to antigen-stimulate the lymphocytes introduced in the second step, In liver tissue! A method comprising the third step of inducing CD4 + FasL + immunoregulatory T cells.

[0016] 2. The method according to item 1, wherein the lymphocyte force CD4-positive T cells obtained in the first step are used.

[0017] 3. The method according to Item 1, wherein in the second step, the lymphocyte is introduced by blood injection and Z or intraperitoneal injection.

[0018] 4. The method according to Item 1, wherein the liver disorder comprises at least one of viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis, and cholangitis.

[0019] 5. The method according to Item 1, wherein the specific antigen is hapten protein.

[0020] 6. Mammal power The method for treatment or prevention according to claim 1, wherein the lymphocyte is specific to the antigen prepared by proliferating and activating the extracted lymphocyte with a specific antigen. Antigen-specific lymphocytes for use in.

[0021] 7. A pharmaceutical composition for treating or preventing liver damage in mammals, comprising CD4 + FasL + immunoregulatory T cells as an active ingredient.

[0022] 8. The pharmaceutical composition according to item 7, wherein the cell secretes IL-4.

[0023] 9. The medical agent according to item 7, wherein the cell is a human CD4 + FasL + immunoregulatory T cell. Drug composition.

The invention's effect

[0024] In the method of the present invention, liver damage can be suppressed in a non-antigen-specific manner by CD4 + FasL + immunoregulatory T cells induced in the liver by oral administration of the antigen. This also makes it possible to handle multiple types of liver damage at the same time. In other words, it is advantageous in that it is not necessary to adopt a different treatment method for each type of liver disorder. In particular, among human hepatitis, there are those for which self-antigen targets, hepatitis virus immunogenic epitopes, etc. have not been elucidated. It is possible to effectively suppress the progress of the failure. That is, even if a pathogenic antigen that causes liver damage cannot be identified, the liver damage can be suppressed by the method of the present invention.

[0025] Further, according to the method of the present invention, it is possible to treat and prevent liver damage using any antigen. That is, since so-called third-party antigens can be used, it is possible to select an antigen that is easily available in large amounts, as compared with conventional self-antigens, and use it as the specific antigen of the present invention. For this reason, appropriate treatment can be performed according to the target patient and the type of liver disorder.

[0026] Furthermore, in the present invention, once lymphocytes are once introduced into the body, liver damage can be suppressed simply by taking a specific antigen from the oral route. That is, since it is easy to take and there is less risk of side effects, the mental and physical burden on the patient can be greatly reduced compared to conventional methods. In addition, since no special medical equipment is required and home treatment is possible, the economic burden can be reduced.

[0027] Even at a stage where liver damage has not yet developed, it is possible to prevent the onset in advance by taking a specific antigen on a daily basis, which is also an effective method for preventing liver damage. is there.

[0028] The method of the present invention having such characteristics can be applied to the treatment of liver disorders in mammals (particularly humans). For example, it can be used to treat various liver disorders such as viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis. Among these, chronic inflammation (chronic hepatitis), especially based on infiltration of Thl lymphocytes It is also suitable for the treatment of various inflammations caused by the above.

Brief Description of Drawings

[0029] FIG. 1 shows suppression of the progression of OVA-specific hepatitis in D011.10 mice administered with OVA. (A) shows the serum levels of AST and ALT. (B) shows the result of H & E staining of a liver specimen. (C) shows proliferative responses in IHLs and splenocytes, IFN-γ secretion and IL-4 secretion level.

FIG. 2 shows suppression of progression of Con-A specific hepatitis in D011.10 mice administered with OVA. (A) shows the levels of AST and ALT in serum. (B) shows the results of observation of liver specimens by H & E staining and TUNEL method. (C) shows the level of proliferative response, IFN-y secretion and IL-4 secretion in IHLs and splenocytes.

FIG. 3 shows that the progression of Con-A-induced hepatitis in BALB / c mice receiving CD4 + T cells from DO11.10 mice administered with OVA is suppressed. (A) shows the levels of AST and ALT in serum. (B) shows the results of observation of liver specimens by H & E staining and TUNEL method. (C) shows the level of proliferative response, IFN-γ secretion and IL-4 secretion in IHLs and splenocytes.

[Fig. 4] Fig. 4 shows the progression of hepatitis induced by Con-A in BALB / c mice transfected with OVA-specific CD4 + T cells from naive DO11.10 mice and orally administered with OVA. It is shown to suppress. (A) shows the levels of AST and ALT in serum. (B) shows the results of observation of liver specimens by H & E staining and TUNEL method. (C) shows the proliferation response of IHLs and splenocytes, the levels of IFN-γ secretion and IL-4 secretion.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] The method of the present invention is a method for non-antigen-specific treatment or prevention of mammalian liver damage,

(3) To the mammal 1) The antigen itself and Z or 2) Equivalent to the antigen U ヽ antigen activity By orally administering a substance having the above, the lymphocytes introduced in the second step are antigen-stimulated and enter the liver tissue! A method comprising the third step of inducing CD4 + FasL + immunoregulatory T cells.

[0031] First step

In the first step, lymphocytes specific to the antigen are prepared by growing and activating lymphocytes removed from the mammal with a specific antigen (hereinafter also referred to as “specific antigen”).

[0032] The method for extracting lymphocytes from mammals is not particularly limited, and any known method may be used. For example, any of a method of collecting from peripheral blood and a method of collecting internal organ force may be used. In particular, a method of collecting peripheral blood is desirable in that the burden on the patient is small and antigen-specific T cells can be efficiently induced in the second step described later. In collecting peripheral blood, for example, peripheral blood of the arm may be collected. The amount of blood collected should be about 10 to 50 mL as long as a predetermined amount of lymphocytes can be obtained.

[0033] The number of lymphocytes to be collected is not particularly limited, and depends on the amount of blood collected as described above. For example, when about 10 to 15 mL of blood is collected, about 10 million lymphocytes can be collected. Separation and collection of lymphocytes can be carried out, for example, by specific gravity centrifugation.

[0034] Next, the lymphocytes specific to the antigen are collected by growing and activating the extracted lymphocytes with the specific antigen. Proliferation and activation methods using specific antigens include known lymphocyte culture methods (eg, Watanabe T, Yoshida M, Shirai Y, Yamori M, Yagita H, Ito T, Chiba T, Kita T, and Wakatsuki Y. Administration). of an antigen at a high dose generates regulatory CD4 + T cells expressing CD95 ligand and secreting IL-4 in the liver. J Immunol 2002; 168: 2188-99). That is, it is acceptable if a culture solution containing a specific antigen is brought into contact with lymphocytes and stimulated. In this case, the concentration of the specific antigen in the culture solution can be appropriately adjusted within a range of usually about 1 to 50 g / mL. Further, for example, a method of culturing a large amount of lymphocytes while adding a culture solution stepwise can be employed. In the present invention, the antigen can be stimulated with a combination of anti-CD3 antibody, IL-2, or the like, if necessary, when proliferating with a specific antigen. [0035] Lymphocytes specific for the antigen can be collected according to a known method. For example, desired lymphocytes can be recovered by specific gravity centrifugation or the like. The antigen-specific lymphocyte is preferably a T cell (particularly a CD4 positive T cell (CD4 + T cell)). If necessary, prior to introduction of lymphocytes into the liver, a sterility test or the like may be performed on the collected lymphocytes in advance.

[0036] The specific antigen is not particularly limited as long as the target mammal does not ingest oral force on a daily basis (so-called pseudoantigen). For example, in the case of mammals other than humans, ovalbumin (Onolevmin; OVA), haptenized proteins, and the like can be used. Moreover, for humans, for example, noh and petit protein can be preferably used.

[0037] Examples of hapten protein include haptenized proteins (peptides) that are eaten on a daily basis. The protein may be either animal protein or vegetable protein. For example, soy protein can be preferably used.

[0038] The type of hapten is not limited, and a known hapten can be selected as appropriate.

Examples include KLH (Keyhole Limpet hemocyanin), picryl chloride, nitrophenol (TNP), azobenzenearsonate (ABA), dinitrochlorbenzene (DNCB), and the like. These can be used alone or in combination of two or more. Using these, a protein or the like may be haptenized according to a known method. Moreover, the haptenized protein can use a commercial item.

[0039] The present invention also includes antigen-specific lymphocytes obtained in the first step. For example, the antigen-specific lymphocyte can be used in the form of 1) the antigen itself and Z or 2) a substance having an antigenic activity equal to that of the antigen and a kit. This kit can be used according to the method from the second step onwards.

[0040] Second step

In the second step, the antigen-specific lymphocytes obtained in the first step are introduced into the liver of the mammal.

[0041] The method for introducing the lymphocyte is not limited. For example, a method of injecting into a vein (infusion), a method of injecting into an artery (eg, hepatic artery), or a method of injecting into the abdominal cavity may be employed. it can. These methods are appropriately determined according to the site of liver injury, the degree of progression of liver injury, etc. can do. In particular, the drip method is preferred because it can reduce the burden on the patient. The infusion method may be the same as the method adopted for general cancer adoptive immunotherapy.

[0042] Further, the lymphocytes are dispersed in, for example, a commercially available culture solution, in a form dispersed in another appropriate solvent or solution, or in a commercially available saline solution for infusion. Alternatively, it may be in a suspended form. In this case, other site force-in and the like can be introduced together with the lymphocytes. The concentration of the lymphocytes in the case of using a solvent or the like can be appropriately set according to the type of liver injury or the like.

[0043] The amount of lymphocytes introduced (per dose) may be sufficient to reach the liver and remain. In general, for adults (about 60 kg), it is desirable to use about 10 ⁵ to 10 ^Ω , especially 10 ⁶ to 10 ⁹ .

[0044] The introduction of the lymphocytes may be performed once, or may be performed twice or more if necessary.

When performing two or more times, it is possible to introduce lymphocytes once a week as one course, and this can be performed several times. These schedules can be set as appropriate according to the type of liver disorder, progress, etc.

[0045] Most of the lymphocytes introduced into the vein or the like in the second step are captured in a predetermined organ (for example, liver, spleen, lung, etc.) by a physiological mechanism. Some of the lymphocytes also reach other organs, but other organs have a much lower chance of encountering antigens than the liver, and are usually deceived by apoptosis.

[0046] Third step

In the third step, the lymphocytes introduced in the second step are orally administered to the mammal 1) the antigen itself and Z or 2) a substance having the same antigenic activity as the antigen. Stimulates in the liver tissue! Induces CD4 + FasL + immunoregulatory T cells.

[0047] To the mammal into which the lymphocyte has been introduced, 1) the antigen itself and Z or 2) a substance having U-antigen activity, such as the antigen, is orally administered. The substance having an antigenic activity equal to that of the antigen is one that induces CD4 + FasL + immunoregulatory T cells in the liver tissue by stimulating the lymphocytes introduced in the second step! If limited However, those having the same antigenic activity as the antigen itself can be appropriately selected and used.

[0048] In oral administration, the time from introduction of the lymphocytes (at the time of the final introduction when introduced multiple times) to oral administration (at the time of the first oral administration) is, for example, the type of liver disorder, patient constitution, etc. Depending on the force, it is generally 24 to 48 hours.

[0049] In the case of oral administration, 1) the antigen itself and Z or 2) substances having antigenic activity such as the antigen (hereinafter 1) 2) are collectively referred to as "specific antigen". The dose depending on the type of the specific antigen can be appropriately set within the range of about 1 to 100 g per day for adults (about 60 kg). The dose can be appropriately changed depending on the degree of progression of liver injury.

[0050] The administration period'administration interval may be set so that the progression of liver injury can be sufficiently suppressed. For example, it may be daily, or it may be continued every other day or every other day for 1 to 12 months.

[0051] When administered orally (orally ingested), the antigen can be formulated according to a known method. For example, tablets, capsules, suspensions, liquid medicines and the like can be mentioned. In this case, known additives such as binders, excipients, fragrances, sweeteners, diluents, and coloring agents can be blended as necessary.

[0052] <Action>

In the present invention, after antigen-specific lymphocytes have been transferred into the body, a specific antigen administered separately orally reaches the liver, whereby CD4 + FasL + immunoregulatory T cells appear in the liver tissue.

[0053] More specifically, when the antigen is orally administered in the presence of antigen-specific lymphocytes, the specific antigen reaches the liver and is presented by CDllc positive cells in the liver sinusoids. . Then, the transferred T cells are stimulated with a specific antigen, and CD4 positive cells expressing FasL appear in the liver. At this time, CD4 positive cells producing INF-γ die, and CD4 positive cells producing IL-10, IL-4, TGF-8, etc. survive. As a result, CD4 + FasL + immunoregulatory T cells secrete these cytodynamic ins and effectively suppress liver damage such as hepatitis by using Fas ligand, which leads to cirrhosis, liver cancer, etc. Progression Can be prevented in advance.

Example

[0054] Examples will be shown below to further clarify the features of the present invention. However,

The scope of the present invention is not limited to the examples.

[0055] <Experimental procedure>

a. Experimental animals

BALB / c mice were purchased from Shizuoka Experimental Animal Research Society (Akira Hamamatsu). D11.10 mouse with transgenic CD4 + T cell receptor for ovalbumin (OVA) 323-329 peptide fragment was provided by KM Murphy (Faculty of Medicine, University of Washington) Murphy KM, Heimberger AB, Loh DY. Induction by antigen of intrathymic apoptosis of CD4 + CD8 + TCRlo thymocytes in vivo. Science 1990; 250: 17 20-3].

[0056] b. Induction of OVA-specific hepatitis

Induction of OVA-specific flames Nishimura T, Ohta A. A crinical role for antigen— sped fic Thl cells in acute liver injury in mice. J. Immunol 1999; 162: 6503— did. A ribosome conjugate containing OVA was prepared by mixing 50 mg / ml OVA (manufactured by Sigma) and Coatsome EL-01-A (manufactured by NOF Corporation). 8 week old D011.10 mice were orally administered with lOOmg OVA or PBS (Phosphate-buffered saline) every other day for a total of 5 times. Three days after the last administration, OVA ribosome solution was administered by intravenous injection of 0.2 ml. After 48 hours, the mice were sacrificed and the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were measured with a transamylase C2 kit (manufactured by Wako Pure Chemical Industries, Ltd.).

[0057] Induction of hepatitis by c-concanavalin A (Con-A)

Oral administration of lOOmg of OVA or PBS (Phosphate-buffered saline) was orally administered every other day to the same 8 week old D011.10 mice as b. Three days after the last administration, Con-A was administered by 0.2 ml intravenous injection. Sixteen hours after the administration, the mice were sacrificed, and the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were changed to transamylase C2 kit (manufactured by Wako Pure Chemical Industries, Ltd.). It was measured by.

[0058] d. Induction of hepatitis by Con-A after adoptive transfer

For 8 weeks old BALB / c mice, 4.7 × 10 ⁷ splenocytes purified from DO11.10 mice (including 4.5 × 10 ⁶ OVA-specific CD4 + T cells) were adoptively transferred. Preparation and transfer of the cells were performed according to ef below.

[0059] Three days after the transfer, lOOmg of OVA or PBS was administered 5 times in total every other day to these transplanted mice. Some of these were D011.10 mice immunized by mouth as described above. Three days after the last dose, 2 mg / ml Con-A (Sigma) was administered intravenously as described in Tiegs et al. Sixteen mice were sacrificed 16 hours after administration.

[0060] e. Purification and adoptive transfer of liver-specific CD4 + T cells

By performing positive selection and negative selection using AutoMACS (manufactured by Miltenyi Biotech), CD4 positive cells and CD4 negative cells such as liver and spleen were purified, respectively. Purity by this method exceeded 90%. BALB / c mice were adoptively transferred with a portion of these cells ( ⁵ × 10 ⁵ per mouse).

[0061] f. Isolation of intrahepatic lymphocytes (IHLs) and splenocytes

IHLs were prepared according to Watanabe T et al. (2002). In other words, after 5 ml of PBS was perfused from the portal vein into the liver, the serum-free RPMI (Rosewell Park Memorial Institute, cell culture medium) containing 0.05% collagenase and 0.002% DNA-degrading enzyme (manufactured by Nakakai Tesque) was used. The cell suspension was digested for 40 minutes at 37 ° C. Hepatocytes were removed by centrifugation at 300 rpm for 3 minutes. IHLs were collected at the interface by density gradient centrifugation at 2000 rpm for 20 minutes using 24% metrizamide.

[0062] Tsukihira Itodao, Kruisbeek AM. Preparation of cell suspension from spleen, thymus, and lymph node. In: Coligan JE, Kruisbbeek AM, Margulies DH, Shevach EM, Stronger W, eds. Current protocols in Immunology. New Prepared according to the method described in York: John Wiery &Sons; 1993. p.3. 1.3.

[0063] g. Growth and site forces

Mouse force IHLs and splenocytes prepared with ConA or OVA-ribosome administration were prepared (previous Note f). These cells (1 X 10 ⁵ / well) were transferred to 1 μg / ml OVA 323-339 peptide or anti-CD3 antibody (Pharmingen) on a U-shaped bottom 96-well culture plate (Sumitomo Betalite). Stimulated with. Cells were cultured for 72 hours and 1 μCi of [ ^{3 3} ] thymidine was added during the last 16 hours. Cells were collected with a multi-sample cell collection device. Thereafter, the amount of introduced thymidine was measured with a top count micro flat plate scintillation counter (manufactured by Packard). Secretion of IFN y and IL-4 in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). For IFN-γ and IL-4, the capture and pyotinylated antibodies were from Pharmigen and Endogen, respectively. Recombinant IFN-γ and IL-4 made by Petrotec were used as an ELISA standard for ELISA.

[0064] h. Analysis

Liver tissue was fixed in 10% buffer formalin phosphate and immersed in paraffin. Thereafter, test specimens were prepared and stained with the hematoxylin and eosin method (H & E). Hepatocellular death due to apoptosis in deparaffinized specimens was determined using the TUNEL method (terminal deoxynucleotidyl of fragmented DNA) using the Apo TACS-DAB In Situ Apoptosis Detection Kit (Trevingen, Gaithersburg, MD). (DUTP-Piotin end labeling with transferase).

[0065] <Reference Example 1>

Administration of an antigen at a high dose generates regulatory CD4 + T cells expressing CD95 ligand and iJ dAVatanabe T, et al. Administration of an antigen at a high dose generates regulatory CD4 + T cells expressing CD95 ligand and secreting IL—4 in the liver. J Immunol 2002; 168 (5): 2188-99.

[0066] In Reference Example 1, it was examined whether CD4 + regulatory T cells suppress the progression of antigen-specific liver injury. Therefore, according to the procedure shown in the above b, the inhibitory effect on liver damage was examined using an OVA-specific hepatitis model mouse obtained by intravenous injection of ribosomes containing OVA.

[0067] FIG. 1 shows the effect of suppressing the progression of OVA-specific hepatitis in D011.10 mice administered with OVA. (A) shows the levels of AST and ALT in serum. (B) shows the result of H & E staining of a liver specimen. Around the portal vein of the liver of mice administered with PBS, the observation of mononuclear cells Significant infiltration was observed (arrow part). (C) shows proliferative response, levels of IFN-γ and IL-4 secretion in IHLs and splenocytes. These measurements followed the methods of f and g. The results are shown as average person SD. These are shown as the results of two independent experiments.

[0068] As shown in FIGS. 1A and B, inoculation of DO11.10 mice receiving PBS with OVA liposomes resulted in significant increases in serum AST and ALT levels in the liver. Infiltration of mononuclear cells in the periportal region was caused extensively. In contrast, serum transamidinase and cell infiltration were not observed in D011.10 mice that received OVA and then received OVA ribosomes. Thus, it can be understood that the progression of liver injury can be almost completely prevented by oral administration of 0 VA before inoculation with OVA ribosome.

[0069] FIG. 1C shows the results of evaluating the force-in-cytogenesis of these mouse force-purified IHLs intrahepatic lymphocytes. As shown in FIG. 1C, in the IHLs of mice administered orally with OVA, the proliferation reaction specific to the OVA peptide or the proliferation reaction specific to the anti-CD3 antibody was remarkably suppressed. In addition, a marked suppression of IFN-γ secretion and a marked increase in IL-4 were observed. Such changes were not observed in the spleen. This shows that OVA-specific hepatitis progression can be suppressed by oral administration of OVA.

[0070] <Reference Example 2>

CD4 + regulatory T cells in the livers of mice that received OVA administration, the antigen non-specifically inhibit the proliferation of T cells (supra Watanabe et al., 2002) _o In addition, as described above, OVA orally administered The proliferative response of IHLs in the given mice was also non-antigen-specifically suppressed.

[0071] In Reference Example 2, it was examined whether or not the progression of non-antigen-specific hepatitis induced by the OVA oral administration power Con-A performed on D011.10 mice could be suppressed. This was done according to the procedure in c above.

FIG. 2 shows the effect of suppressing the progression of Con-A specific hepatitis in D011.10 mice administered with OVA. (A) shows AST and ALT levels in serum. (B) shows the results of observation of liver specimens by H & E staining and TUNEL method. According to the results of TUNEL observation of the liver specimens of mice administered with PBS, many lesions due to hepatocyte death were confirmed (arrows). (C) shows the level of proliferative response, IFN-γ secretion and IL-4 secretion in IHLs and splenocytes. These measurements followed the methods of f and g. The average person SD It shows with. These are shown as the results of two independent experiments.

[0073] As shown in FIGS. 2A and 2B, oral administration of OVA before Con-A inoculation significantly suppressed the increase in serum ALT and AST. In the liver of mice that received PBS orally, massive death of hepatocytes was confirmed by H & E staining or TUNEL. In contrast, TUNEL + cells showed little force in the livers of mice that received oral OVA. This indicates that histologically hepatocyte damage due to hepatitis can be prevented.

[0074] As shown in FIG. 2C, in the mice that received OVA orally, the proliferation response by IHLs and the secretion of IFN-γ were markedly decreased and the secretion of IL-4 was increased. Thus, it can be seen that the hepatitis induced by Con-A can be suppressed by oral administration of OVA.

[0075] <Reference Example 3>

In Reference Example 3, it was investigated whether adoptive transfer of CD4 + T cells into the liver could suppress Con-A-induced hepatitis. CD4 + T cells were purified from the liver and spleen of D011.10 mice that received oral OVA, transferred to BALB / c mice, and then inoculated with Con-A. This was done according to the procedure in d above.

FIG. 3 shows that the progression of Con-A-induced hepatitis in BALB / c mice that received CD4 + T cells from D011.10 mice administered with OVA was suppressed. (A) shows the levels of AST and ALT in serum. (B) shows the results of observation of liver specimens by H & E staining and TUNEL method. The force of DO11.10 mice to which PBS was administered was also confirmed by the TUNEL method of liver specimens in mice transfected with CD4 + T hepatocytes, and many lesions due to hepatocyte death were confirmed (arrow part). (C) shows the level of proliferative response, IFN-γ secretion and IL-4 secretion in IHLs and splenocytes. These measurements followed the methods of f and g. The result is shown as average person SD. These are shown as the results of two independent experiments.

[0077] As shown in Figs. 3A and B, BALB / c mice receiving CD4 + T cells from the liver of DO11.10 mice that were orally administered OVA! Being! / In other groups of mice, elevated serum AST and ALT and massive hepatocyte death were observed. The inhibitory effects of adoptively transferred CD4 + regulatory T cells were associated with local Th2 responses.

[0078] As shown in Fig. 3C, CD4 + T cells in the liver of DO11.10 mice administered orally with OVA were received. Only in mice, marked suppression of IFN-γ secretion and proliferative responses was observed. In contrast, IL-4 secretion by IHLs was markedly increased. These suppressive effects were not seen in mice that received Con-A after adoptive transfer of CD4 negative cells.

[0079] <Example 1>

The present inventor has previously shown that OVA-specific naive CD4 + T cells differentiate into FasL + regulatory T cells by oral administration of OVA using DO11.10 mice.

[0080] In Example 1, it was examined whether oral progression of OVA could suppress the progression of Con-A-induced hepatitis in the presence of a certain amount of OVA-specific CD4 + T cells. DO11.10 mice were given PBS or OVA 5 times every other day according to b above. Then, according to e, B ALB / c mice were adoptively transferred with 4.7 × 10 ⁷ splenocytes purified from D011.10 mice (including 4.5 × 10 ⁶ OVA-specific CD4 + cells). After 3 days of transfer, these mice were inoculated with 0.2 mg intravenously of 2 mg / ml Con-A. Mice were sacrificed 16 hours after Con-A inoculation.

[0081] Fig. 4 shows Con-A-induced hepatitis in BALB / c mice transfected with naive DO11.10 mice and orally administered OVA-specific CD4 + T cells! Demonstrate the effect of suppressing progress. (A) shows the serum levels of AST and ALT. (B) shows the results of observation of liver specimens by H & E staining and TUNEL method. Lymphocyte death was observed in many areas of liver specimens from mice receiving PBS. (C) shows proliferation response, IFN-γ and IL-4 levels in IHLs and splenocytes. These measurements followed the methods of f and g. Results are shown as average person SD. These are shown as independent experimental results.

[0082] As shown in FIGS. 4A and B, it can be seen that oral administration of OVA suppresses the increase in AST and ALT and suppresses the massive death of hepatocytes.

[0083] As shown in Fig. 4C, mice that were orally administered OVA after being transfected with OVA-specific CD4 + T cells showed increased secretion of IL-4 as well as suppression of IFN-γ secretion and proliferative response. It was.

[0084] Thus, hepatitis caused by an antigen different from the antigen (that is, Con--) by transferring CD4 + T cells specific for a specific antigen (here OVA) and then ingesting the antigen also by oral force. It can be seen that hepatitis induced by A) can also be suppressed. That is, the course of a specific antigen It can be seen that oral administration generates CD4 + regulatory T cells, which can suppress the development of liver damage in a non-antigen-specific manner.

[0085] From these results, peripheral blood force lymphocytes of the patient were collected in advance, antigen-specific sputum cells were prepared in vitro, then returned to the patient's body, and the same antigen as described above was eaten. By doing so, it can be seen that treatment or prevention of hepatitis and the like becomes possible.

[0086] From these facts, CD4 + FasL + immunoregulatory T cells can be expected as an active ingredient of a pharmaceutical composition for treating liver injury.

Claims

Claims [1] A method for non-antigen-specific treatment or prevention of mammalian liver damage,

Or In liver tissue! A method comprising the third step of inducing CD4 + FasL + immunoregulatory T cells.

[2] The method according to claim 1, wherein the lymphocytes obtained in the first step are CD4-positive T cells.

[3] In the second step, the lymphocyte is introduced by blood injection and Z or intraperitoneal injection.

The method of claim 1.

[4] The method according to claim 1, wherein the hepatic disorder comprises at least one of viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis and cholangitis.

[5] The method of claim 1, wherein the specific antigen is a hapten protein.

[6] A lymphocyte specific to the antigen prepared by growing and activating lymphocytes taken from a mammal with a specific antigen, which is used for the treatment or prevention method according to claim 1. For antigen-specific lymphocytes.

[7] A pharmaceutical composition for treating or preventing liver damage in mammals, comprising CD4 + FasL + immunoregulatory T cells as an active ingredient.

8. The pharmaceutical composition according to claim 7, wherein the cell secretes IL-4.

[9] The pharmaceutical composition according to claim 7, wherein the cell is a human CD4 + FasL + immunoregulatory T cell.