JP2007320852A - Method of preventing or treating of hepatopathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simpler method of preventing or treating hepatopathy which exhibits relatively few side effects. <P>SOLUTION: The method of antigen-nonspecifically preventing or treating mammalian hepatopathy comprises (1) a first step of proliferating and activating lymphocytes taken out from a mammal by a specific antigen to prepare lymphocytes which are specific to the antigen, (2) a second step of introducing the antigen-specific lymphocytes obtained in the first step into the liver of the mammal and (3) a third step of orally administering (i) the antigen itself and/or (ii) a substance which is equivalent in antigenic activity to the antigen to the mammal to stimulate the lymphocytes, which have been introduced in the second step, with the antigen, thereby inducing CD4+FasL+immunomodulatory T cells in the liver tissue. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for preventing or treating liver disease. More specifically, the present invention relates to a method for preventing or treating liver damage by utilizing immunological anti-inflammatory reaction by orally administering a T cell transfer and an antigen specific to the transferred T cell.

  Hepatitis and the like in mammals, especially humans, are diseases that can lead to cirrhosis, liver cancer, and death. Liver damage is caused not only by hepatitis due to viral infection, but also by an immune reaction against self antigens expressed in the liver.

  Today, patients with viral hepatitis and autoimmune liver disease are treated with antiviral agents or immunosuppressants such as interferons, reverse transcriptase inhibitors, steroid hormones, and the like. However, these drug treatments are not necessarily treatments based on cell mechanisms causing liver damage, and the therapeutic effects are not sufficient, and side effects cannot be ignored.

  By administering the antigen orally, T cells (suppressor T cells, regulatory T cells) that suppress immune inflammatory reactions are induced in the body. As the induction mechanism, when a large amount of antigen is administered, antigen-specific T cells are removed (clone removal theory). On the other hand, when a small amount of antigen is administered, it is considered that regulatory T cells are induced in the intestinal lymph nodes (Peier's plate).

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

  From an anatomical point of view, portal tolerance, which is established when antigenic substances derived from food and gut microbiota are constantly sent to the liver via the portal vein, is involved in the tolerance-inducing properties of the liver. (Crispe IN, Dao T, Klugewitz K, Mehal WZ, Metz DP. The liver as a site of T-cell apoptosis: graveyard, or killing field? Immunol Rev 2000; 174: 47-62). An example of an application of this characteristic of easily inducing immune tolerance inherent in the liver is, for example, islet transplantation from the portal vein in the treatment of insulin-dependent diabetes (Shapiro, AM et al., N. Engl. J. Med. 343.230 (2000)).

  Recently, various attempts have been made to prevent inflammation by inducing immune tolerance by orally administering a self-antigen, which is thought to cause each disease, to a patient. For example, rheumatoid arthritis (Trentham, DE et al. Science, 261: 1727 (1993)), multiple sclerosis (Weiner, Hlet al., Science, 2589: 1321 (1993)), type 2 diabetes (Lalej- Bennis D et al. Efficacy and tolerance of intranasal insulin administered during 4 months in severely hyperglycaemic Type 2 diabetic patients with oral drug failure: a cross-over study.Diabet Med 2001; 18 (8): 614-8.), Uvea Treatment of patients with inflammation (Thurau SR et al. Oral tolerance for treating uveitis-new hope for an old immunological mechanism.Prog Retin Eye Res 2002; 21 (6): 577-89) orally Has been done.

  However, these treatment methods require autoantigens, but it is difficult to obtain and secure a sufficient amount of autoantigens for treatment.

  In addition, since these have various immune tolerance mechanisms (Weiner HL. Oral tolerance, an active immunologic process mediated by multiple mechanisms.J Clin Invest 2000; 106 (8): 935-7) Cells with an inhibitory effect cannot be induced efficiently.

  In addition, these methods may in some cases exacerbate the disease or induce another disease (Blanas E, Heath WR.Oral administration of antigen can lead to the onset of autoimmune disease.Int Rev Immunol 1999; 18 (3): 217-28).

  Thus, since there is no effective treatment other than liver transplantation for patients with severe hepatic dysfunction due to advanced hepatitis, development of new treatments or prevention methods for liver disorders such as hepatitis is hoped for. It is rare.

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

  In view of the problems of the prior art as described above, the present inventor has found that the above object can be achieved by a method comprising a specific process as a result of intensive studies, and has completed the present invention.

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

1. A method for non-antigen-specific treatment or prevention of mammalian liver damage, comprising:
(1) a first step of preparing lymphocytes specific for the antigen by proliferating and activating lymphocytes taken from the mammal with the specific antigen;
(2) The second step of introducing the antigen-specific lymphocyte obtained in the first step into the liver of the mammal, and (3) 1) the antigen itself and / or 2) the same as the antigen. The method comprises a third step of orally administering a substance having antigenic activity to stimulate the lymphocytes introduced in the second step, and induce CD4 + FasL + immunoregulatory T cells in liver tissue. And how to.

  2. Item 2. The method according to Item 1, wherein the lymphocyte obtained in the first step is a CD4-positive T cell.

  3. Item 2. The method according to Item 1, wherein in the second step, introduction of the lymphocyte is performed by blood injection and / or intraperitoneal injection.

  4). Item 2. The method according to Item 1, wherein the liver disorder comprises at least one of viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis and cholangitis.

  5. Item 2. The method according to Item 1, wherein the specific antigen is a haptenized protein.

  6). A lymphocyte specific for the antigen prepared by growing and activating lymphocytes taken from a mammal with a specific antigen, the antigen for use in the treatment or prevention method according to claim 1 Specific lymphocytes.

  In the method of the present invention, non-antigen-specific liver damage can be suppressed by CD4 + FasL + immunoregulatory T cells induced in the liver by oral administration of the antigen. Thereby, it is also possible to cope with a plurality of types of liver disorders at the same time. In other words, it is advantageous in that it is not necessary to separately take a different treatment method for each type of liver disorder. In particular, among human hepatitis, there are those in which the self-antigen target, hepatitis virus immunogenic epitope, etc. have not yet been elucidated. Progress can be effectively suppressed. That is, even if a pathogenic antigen that causes liver damage cannot be identified, the method according to the present invention can suppress the liver damage.

  Moreover, according to the method of the present invention, it is possible to treat or prevent liver damage using any antigen. That is, since a so-called third-party antigen can be used, an antigen that is easily available in large quantities can be selected and used as the specific antigen of the present invention instead of the conventional autoantigen. For this reason, it is possible to perform an appropriate treatment corresponding to the target patient, the type of liver disorder, and the like.

  Furthermore, in the present invention, once lymphocytes are once introduced into the body, liver damage can be suppressed by simply taking a specific antigen from the oral route. That is, since the intake is easy and the risk of side effects is low, the mental and physical burden on the patient can be greatly reduced as compared with the conventional method. Further, since no special medical equipment is required and home treatment is possible, the economic burden can be reduced.

  In addition, even in a stage where hepatic disorder 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 effective as a method for preventing hepatic disorder.

  The method of the present invention having such characteristics can be used for the treatment of various liver disorders such as viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis. . Among these, it is also suitable for the treatment of chronic inflammation (chronic hepatitis), particularly various inflammations caused by infiltration of Th1-type lymphocytes.

The method of the present invention is a method for non-antigen-specific treatment or prevention of mammalian liver damage,
(1) a first step of collecting lymphocytes specific to the antigen by growing and activating lymphocytes taken from the mammal with the specific antigen;
(2) The second step of introducing the antigen-specific lymphocytes obtained in the first step into the mammal into the liver, and (3) The antigen is introduced into the mammal in the second step by oral administration. And a third step of inducing CD4 + FasL + regulatory T cells in liver tissue by further stimulating the lymphocytes with antigen.

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

  The method for extracting lymphocytes from the mammal is not particularly limited, and any known method may be followed. For example, any of a method of collecting from peripheral blood and a method of collecting from a body organ 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 may be collected from an arm vein. The amount of blood collected may be such that a predetermined amount of lymphocytes can be obtained, and usually about 10 to 50 mL.

  The number of lymphocytes to be collected is not particularly limited, and depends on the amount of collected blood. For example, when about 10 to 15 mL of blood is collected, about 10 million lymphocytes can be collected. Lymphocytes can be separated and collected by, for example, specific gravity centrifugation.

  Next, the lymphocytes specific to the antigen are collected by growing and activating the extracted lymphocytes with the specific antigen. Proliferation and activation with a specific antigen can be performed by 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 Imminol 2002; 168: 2188-99). That is, a culture solution containing a specific antigen may be stimulated by contacting with lymphocytes. 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 μg / 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, when proliferating and activating with a specific antigen, antigen stimulation can also be performed by using an anti-CD3 antibody, IL-2 or the like, if necessary.

  Collection of lymphocytes specific for the antigen can be performed 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, a sterility test or the like may be performed on the collected lymphocytes in advance prior to introduction of lymphocytes into the liver.

  The specific antigen is not particularly limited and may be any antigen that is not routinely ingested by the target mammal (so-called pseudoantigen). For example, in the case of mammals other than humans, ovalbumin (Ovalbumin; OVA), haptenized proteins, and the like can be used. For humans, for example, haptenized proteins can be preferably used.

  Examples of haptenized proteins 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.

  The type of hapten is not limited and can be appropriately selected from known haptens. For example, KLH (Keyhole Limpet hemocyanin), picryl chloride, nitrophenol (TNP), azobenzenearsonate (ABA), dinitrochlorbenzene (DNCB) and the like can be mentioned. These can be used alone or in combination of two or more. What is necessary is just to haptenize protein etc. according to a well-known method using these. Moreover, the haptenized protein can also use a commercial item.

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

  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 (such as a hepatic artery), a method of injecting into the abdominal cavity, or the like can be employed. These methods can be appropriately determined according to the site of liver injury, the degree of progression of liver injury, and the like. In particular, the drip method is preferable in that the burden on the patient can be reduced. The infusion method may be the same as the method employed in general cancer adoptive immunotherapy and the like.

  The lymphocytes are dispersed or suspended in, for example, a commercially available saline solution in addition to a form dispersed in a commercially available culture solution, a form dispersed in another appropriate solvent or solution, and the like. It may be a different form. In this case, other cytokines 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.

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

  The introduction of the lymphocytes may be performed once, or may be performed twice or more as necessary. When performing two or more times, for example, lymphocyte introduction once a week is considered as one course, and this can be performed several times. These schedules can be appropriately set according to the type of liver disorder, the progress status, and the like.

  Most of the lymphocytes introduced from 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.

Third Step In the third step, 1) the antigen itself and / or 2) a substance having an antigen activity equal to that of the antigen is orally administered to the mammal, whereby the lymphocytes introduced in the second step are treated with the antigen. Stimulates and induces CD4 + FasL + immunoregulatory T cells in liver tissue.

  1) The antigen itself and / or 2) a substance having an antigen activity equal to that of the antigen is orally administered to the mammal into which the lymphocyte has been introduced. The substance having the same antigenic activity as the antigen is not particularly limited as long as it stimulates the lymphocytes introduced in the second step to induce CD4 + FasL + immunoregulatory T cells in the liver tissue. Those having the same antigenic activity as the antigen itself can be appropriately selected and used.

  In oral administration, the time from introduction of the lymphocytes (at the time of the last introduction when introduced multiple times) to oral administration (at the time of the first oral administration) varies depending on, for example, the type of liver disorder, the patient's constitution, etc. Generally, it may be about 24 to 48 hours.

  In the case of oral administration, 1) the antigen itself and / or 2) substances having the same antigenic activity as the antigen (hereinafter, 1) 2) are collectively referred to as “specific antigen”. The dose of) depends on the type of specific antigen and the like, but can be appropriately set within the range of about 1 to 100 g per day for an adult (about 60 kg). The dose can be appropriately changed according to the degree of progression of liver injury.

  The administration period and administration interval may be set so that the progression of liver injury can be sufficiently suppressed. For example, it may be daily, or may be continuously administered for 1 to 12 months every other day or every two or more days.

  In the case of oral administration (oral intake), 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 a binder, an excipient, a fragrance, a sweetener, a diluent, and a colorant may be blended as necessary.

<Action>
In the present invention, after antigen-specific lymphocytes are transferred into the body, specific orally administered antigens reach the liver, and CD4 + FasL + immunoregulatory T cells appear in the liver tissue.

  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 CD11c positive cells in the liver sinusoid. 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 are killed, and CD4 positive cells producing IL-10, IL-4, TGF-β, etc. survive. As a result, CD4 + FasL + immunoregulatory T cells secrete these cytokines and also effectively suppress liver damage such as hepatitis by the action of Fas ligand, which leads to progression to liver cirrhosis, liver cancer, etc. It can be prevented in advance.

  Examples are given below to further clarify the features of the present invention. However, the scope of the present invention is not limited to the examples.

<Experimental procedure>
a. Experimental animals
BALB / c mice were purchased from the Shizuoka Experimental Animal Research Society (Hamamatsu City). The D11.10 mouse carrying the transgenic CD4 + T cell receptor for the ovalbumin (OVA) 323-329 peptide fragment used was provided by KM Murphy (Faculty of Medicine, School 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: 1720-3].

b. Induction of OVA-specific hepatitis
Induction of OVA-specific hepatitis was carried out as described in Nishimura T, Ohta A. Acrical role for antigen-specific Th1 cells in acute liver injury in mice. J. Immunol 1999; 162: 6503-9. OVA-containing liposome conjugate was prepared by mixing 50 mg / ml OVA (Sigma) and Coatsome EL-01-A (Nippon Yushi Co., Ltd.). 100 mg OVA or PBS (Phosphate-buffered saline) was orally administered every other day to DO11.10 mice of 8 weeks after birth for a total of 5 times. Three days after the last administration, OVA liposome solution was administered by 0.2 ml intravenous injection. Mice were sacrificed 48 hours after the administration, 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.).

c. Induction of hepatitis by Concanavalin A (Con-A) A 100 mg OVA or PBS (Phosphate-buffered saline) is added every other day to DO11.10 mice of the same age 8 weeks as b. A total of 5 times oral administration. Three days after the last administration, Con-A was administered by 0.2 ml intravenous injection. Sixteen hours after this administration, 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.).

d. Induction of hepatitis by adopting Con-A after adoptive transfer For 8 weeks old BALB / c mice, 4.7 × 10 ⁷ splenocytes purified from DO11.10 mice (4.5 × OVA-specific CD4 + T cells) 10 including ^six.) was the adoptive transfer. The cells were prepared and transferred according to ef below.

  Three days after the transfer, 100 mg of OVA or PBS was administered to the transplanted mice every other day for a total of 5 times. Some of these were orally immunized with DO11.10 mice as described above. Three days after the last dose, 2 mg / ml Con-A (Sigma) was administered intravenously as described in Tiegs et al. Mice were sacrificed 16 hours after dosing.

e. Purification and adoptive transfer of liver-specific CD4 + T cells
By performing positive selection and negative selection using AutoMACS (Miltenyi Biotech), CD4 positive cells and CD4 negative cells were purified from the liver and spleen, respectively. The purity by this method exceeded 90%. A portion of this cell (5 × 10 ⁵ per mouse) was adoptively transferred to BALB / c mice.

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) 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 metrizamide 24%.

  Spleen cells are from 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 York: John Wiery &Sons; 1993. Prepared according to the method described in p.3.1.3.

g. Proliferation and cytokine assays
IHLs and splenocytes were prepared from mice receiving ConA or OVA-liposome (f). These cells (1 × 10 ⁵ / well) were stimulated with 1 μg / ml of OVA 323-339 peptide or anti-CD3 antibody (Pharmingen) in a U-shaped bottom 96-well culture plate (Sumitomo Bakelite). Cells were cultured for 72 hours and 1 μCi [ ³ H] thymidine was added for 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 IFNγ and IL-4 in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). For IFN-γ and IL-4, the capture and biotinylated antibodies were from Pharmigen and Endogen, respectively. Recombinant IFN-γ and IL-4 manufactured by Petrotec were used as assay standards for the ELISA method.

h. Analysis The liver tissue was fixed in 10% buffer formalin phosphate and immersed in paraffin. Thereafter, a test piece was prepared and stained by the hematoxylin-eosin method (H & E). Hepatocellular death due to apoptosis of deparaffinized specimens was determined by TUNEL method using apoTACS-DAB In Situ Apoptosis Detection Kit (Trevingen, Gaithersburg, MD). End labeling).

<Reference Example 1>
It is said that Watanabe T, et al. Administration of an antigen at a high dose generates regulatory CD4 + T cells expressing CD95 ligand and secreting when the antigen is orally administered to DO11.10 mice administered with OVA, CD4 + regulatory T cells appear in the liver. IL-4 in the liver. J Immunol 2002; 168 (5): 2188-99.

  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, an inhibitory effect on liver damage was examined using an OVA-specific hepatitis model mouse obtained by intravenous injection of liposomes containing OVA.

  FIG. 1 shows the effect of suppressing the progression of OVA-specific hepatitis in DO11.10 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. Remarkable infiltration of mononuclear cells was observed around the portal vein of the liver of mice administered with PBS (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 mean ± SD. These are shown as the results of two independent experiments.

  As shown in FIGS. 1A and B, inoculation of PBS11-treated DO11.10 mice with OVA liposomes resulted in a significant increase in serum AST and ALT, as well as the periportal region in the liver. Infiltration of mononuclear cells was extensively caused. In contrast, serum transamidinase and cell infiltration were not observed in DO11.10 mice that received OVA administration and then received OVA liposomes. Thus, it can be seen that the progression of liver damage can be almost completely prevented by oral administration of OVA before inoculation of OVA liposomes.

  FIG. 1C shows the results of evaluation of cytokine production of IHLs intrahepatic lymphocytes purified from these mice. 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 the progression of OVA-specific hepatitis can be suppressed by oral administration of OVA.

<Reference Example 2>
CD4 + regulatory T cells in the liver of mice receiving OVA suppress T cell proliferation in a non-antigen-specific manner (Watanabe et al., 2002). Further, as described above, the proliferation reaction of IHLs in mice that were orally administered OVA was also suppressed in a non-antigen-specific manner.

  In Reference Example 2, it was examined whether oral administration of OVA made to DO11.10 mice can suppress the progression of antigen-specific hepatitis induced by Con-A. This was performed according to the procedure of c above.

  FIG. 2 shows the inhibitory effect on the progression of Con-A specific hepatitis in DO11.10 mice administered with OVA. (A) shows the levels of AST and ALT in serum. (B) shows the observation results of liver specimens by H & E staining and TUNEL method. According to the observation result of the liver specimen of the mouse administered with PBS by the TUNEL method, 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 results are shown as mean ± SD. These are shown as the results of two independent experiments.

  As shown in FIGS. 2A and 2B, oral administration of OVA before inoculation of Con-A significantly suppressed the increase in serum ALT and AST. In the livers of mice receiving PBS orally, massive death of hepatocytes was confirmed by H & E staining or TUNEL method. In contrast, only a few TUNEL + cells were observed in the livers of mice that were orally administered OVA. This shows that hepatocyte damage due to hepatitis can be prevented histologically.

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

<Reference Example 3>
In Reference Example 3, it was examined whether Con-A-induced hepatitis can be suppressed by adoptive transfer of CD4 + T cells to the liver. CD4 + T cells were purified from the liver and spleen of DO11.10 mice that were orally administered OVA, transferred to BALB / c mice, and then inoculated with Con-A. This was performed according to the procedure of d above.

  FIG. 3 shows that the progression of Con-A induced hepatitis is suppressed in BALB / c mice that received CD4 + T cells from DO11.10 mice administered with OVA. (A) shows the levels of AST and ALT in serum. (B) is the observation result of the liver specimen by H & E staining and TUNEL method. According to the results of the TUNEL method of liver specimens in mice transfected with CD4 + T hepatocytes from DO11.10 mice administered with PBS, numerous 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 results are shown as mean ± SD. These are shown as the results of two independent experiments.

  As shown in FIGS. 3A and B, Con-A-induced hepatitis was significantly suppressed only in BALB / c mice that received liver CD4 + T cells of DO11.10 mice that were orally administered OVA. In other groups of mice, elevated serum AST and ALT and massive hepatocyte death were observed. The inhibitory effect of adoptively transferred CD4 + regulatory T cells was associated with a local Th2 response.

  As shown in FIG. 3C, significant suppression of IFN-γ secretion and proliferative response was observed only in mice that received CD4 + T cells from the liver of DO11.10 mice administered orally by OVA. In contrast, IL-4 secretion by IHLs was markedly increased. These inhibitory effects were not seen in mice that received Con-A after adoptive transfer of CD4 negative cells.

<Example 1>
The 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.

In Example 1, it was examined whether or not the progression of Con-A-induced hepatitis can be suppressed by oral administration of OVA when a certain amount of OVA-specific CD4 + T cells are present. DO11.10 mice were given PBS or OVA 5 times every other day according to b. Subsequently, according to the above e, 4.7 × 10 ⁷ splenocytes purified from DO11.10 mice (including 4.5 × 10 ⁶ OVA-specific CD4 + cells) were adoptively transferred to BALB / c mice. Three days after this transfer, these mice were inoculated with 2 mg / ml of Con-A through a 0.2 ml vein. Mice were sacrificed 16 hours after Con-A inoculation.

  FIG. 4 shows the inhibitory effect on the progression of hepatitis induced by Con-A in BALB / c mice, in which OVA-specific CD4 + T cells were transferred from naive DO11.10 mice and OVA was orally administered. (A) shows serum levels of AST and ALT. (B) shows the observation result of the liver specimen by H & E staining and TUNNEL method. Lymphocyte death was observed in many areas of the liver specimens of mice receiving PBS. (C) shows proliferative response, IFN-γ and IL-4 levels in IHLs and splenocytes. These measurements followed the methods of f and g. Results are shown as mean ± SD. These are shown as independent experimental results.

  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.

  As shown in FIG. 4C, in mice to which OVA-specific CD4 + T cells were transferred and then orally administered with OVA, an increase in IL-4 secretion was confirmed along with suppression of IFN-γ secretion and proliferative response.

  Thus, hepatitis (ie, induced by Con-A) due to an antigen different from the antigen by ingesting the antigen orally after transferring CD4 + T cells specific for a specific antigen (here OVA) It can be seen that hepatitis) can also be suppressed. That is, it is understood that CD4 + regulatory T cells are generated by oral administration of a specific antigen, and these cells can suppress the occurrence of liver damage in a non-antigen-specific manner.

  From these results, by collecting lymphocytes from the patient's peripheral blood in advance, creating antigen-specific T cells in vitro, returning them to the patient's body, and eating the same antigen as above It can be seen that treatment or prevention of hepatitis and the like is possible.

FIG. 1 shows inhibition of the progression of OVA-specific hepatitis in DO11.10 mice administered with OVA. (A) shows serum levels of AST and ALT. (B) shows the result of H & E staining of a liver specimen. (C) shows the level of proliferative response, IFN-γ secretion and IL-4 secretion in IHLs and splenocytes. FIG. 2 shows suppression of progression of Con-A specific hepatitis in DO11.10 mice administered with OVA. (A) shows the levels of AST and ALT in serum. (B) is the observation result of the liver specimen 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. 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) is the observation result of the liver specimen 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 shows that the progression of hepatitis induced by Con-A is suppressed in BALB / c mice transfected with OVA-specific CD4 + T cells from naive DO11.10 mice and orally administered with OVA. (A) shows the levels of AST and ALT in serum. (B) shows the observation results of the liver specimen by H & E staining and TUNEL method. (C) shows the proliferative response of IHLs and splenocytes, levels of IFN-γ secretion and IL-4 secretion.

Claims (6)

A method for non-antigen-specific treatment or prevention of mammalian liver damage, comprising:
(1) a first step of preparing lymphocytes specific for the antigen by proliferating and activating lymphocytes taken from the mammal with the specific antigen;
(2) The second step of introducing the antigen-specific lymphocyte obtained in the first step into the liver of the mammal, and (3) 1) the antigen itself and / or 2) the same as the antigen. The method comprises a third step of orally administering a substance having antigenic activity to stimulate the lymphocytes introduced in the second step, and induce CD4 + FasL + immunoregulatory T cells in liver tissue. And how to. The method according to claim 1, wherein the lymphocytes obtained in the first step are CD4-positive T cells. The method according to claim 1, wherein in the second step, the introduction of the lymphocyte is performed by blood injection and / or intraperitoneal injection. The method according to claim 1, wherein the liver disorder comprises at least one of viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis and cholangitis. 2. The method of claim 1, wherein the specific antigen is a haptenized protein. A lymphocyte specific for the antigen prepared by growing and activating lymphocytes taken from a mammal with a specific antigen, the antigen for use in the treatment or prevention method according to claim 1 Specific lymphocytes.

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