CN1395490A - Antigen-specific IgE antibody production inhibitors - Google Patents

Abstract

The present invention relates to mammalian (including human) IgE antibody production inhibitors and immunomodulatory compositions containing as the active ingredient lactoferrin (LF) or its hydrolyzate; and the invention also relates to IFN- gamma production potentiators containing LF or its hydrolyzate and ceramides.

Description

Antigen-specific IgE antibody production inhibitors

technical field

The present invention relates to IgE production inhibitors which are effective in preventing or improving allergic diseases such as atopic dermatitis. The present invention also relates to compositions for stimulating the immune system of a living body to enhance immunity.

Background technique

Lactoferrin (LF) is an iron-binding glycoprotein that is present not only in milk but also in exocrine fluids such as saliva, tears, bile and pancreatic juice. It can also be released by activated neutrophils at sites of inflammation.

LF has a broad spectrum of biological activities. For example, it includes antimicrobial activity, antiviral activity, antitumor activity, cancer metastasis inhibitory activity and the like.

In addition, LF affects cytokine secretion. For example, LF promotes the secretion of interleukin 8 (IL-8) by human neutrophils, controls the secretion of IL-1, IL-6 and TNFα, and inhibits the expression of GM-CSF gene level.

Selective production of cytokines plays protective or etiological roles in various diseases. For example, most allergies are type I allergies, which are associated with IgE antibodies. The IgE production is induced by a process involving IL-4, on the other hand, the IgE production is controlled by a process involving interferon-gamma (IFN-gamma). The production of IFN-γ is induced by its epistatic IL-12 and IL-18.

IFN-γ also exhibits antiviral, antimicrobial, and antitumor activities that are dependent on various immunomodulatory functions (J. Immunol., 130, 2011-2013, 1983; National Academy of Sciences, USA Journal (Proc. Natl. Acad. Sci. USA), 85, 4874-4878, 1988).

Nakajima et al. reported that when mouse splenocytes were cultured in the presence of a mitogen (Con A) following oral administration of bovine LF (bLF), the production of IFN-γ, but not IL-4, was increased (BioMed Research (Biomedical Research), 29(1), 27-33, 1999).

As mentioned above, LF is involved in the production and regulation of various biological activities and cytokines. The object of the present invention is therefore to reveal new biological activities of LF, and based on this activity, to provide medicaments and functional foods containing LF as an active ingredient. Another object of the present invention is to find an orally ingestible substance which, when combined with LF, has the effect of increasing the biological activity of LF.

Disclosure of the invention

The present inventors found that when mice ingested orally ingested LF, heat-treated LF, or LF hydrolyzate were immunized with ovalbumin (OVA), a major food allergen, the production of OVA-specific IgE antibodies was regulated. Inhibition, the production of antigen-specific IL-4 in the splenocyte culture system was also inhibited. They also found that when the macrophages were cultured in the presence of LF, the production of IL-12 by the macrophages increased. In addition, they also found that the combination of LF and ceramide significantly increased the production of IFN-γ in the splenocyte culture system. Oral ingestion of LF is thought to inhibit the production of antigen-specific IgE antibodies by suppressing the activation of IgE antibody-producing B lymphocytes by inhibiting IL-4 production, and by increasing the production of IL-12 towards Th1 in the Th1/Th2 balance. Caused by dominant movement. That is, oral intake of LF can prevent the reduction of cellular immunity by shifting to Th2 response, and can prevent or improve nephritis caused by allergic disease or antibody reaction. The combination of LF and ceramide significantly increased the production of IFN-γ, suggesting that the combination of LF and ceramide is effective against viral diseases, bacterial infections or tumors.

Specifically, the present invention provides a mammalian (including human) IgE antibody production inhibitor comprising LF or its hydrolyzate as an active ingredient.

The present invention also provides a composition for controlling immunity by inhibiting the production of IgE antibodies in mammals (including humans), which comprises the active ingredient LF or its hydrolyzate.

The present invention also provides an IFN-γ production potentiator composition, an antiviral agent composition, an antibacterial agent composition or an antitumor agent composition for use in mammals including humans, which comprises LF or its hydrolyzate and neural amides.

The present invention also provides an immunopotentiator composition for mammals including humans, comprising LF or its hydrolyzate and ceramide.

The present invention also provides the use of LF or its hydrolyzate for preparing inhibitors of IgE antibody production in mammals (including humans).

The present invention also provides the use of LF or its hydrolyzate for preparing a composition for controlling immunity by inhibiting the production of IgE antibodies in mammals (including humans).

The present invention also provides LF or its hydrolyzate and ceramide for the preparation of IFN-γ production synergist composition, antiviral agent composition, antibacterial agent composition or antitumor agent combination for mammals including humans use of things.

In addition, the present invention also provides the use of LF or its hydrolyzate and ceramide for preparing an immunopotentiator composition for mammals including humans.

The present invention also provides a method for inhibiting IgE antibody production in mammals including humans, comprising administering an effective amount of LF or a hydrolyzate thereof.

The present invention also provides a method of modulating IgE antibody production in mammals including humans, comprising administering an effective amount of LF or a hydrolyzate thereof.

The present invention also provides a method for increasing IFN-γ production in mammals including humans, which comprises administering an effective amount of LF or its hydrolyzate and ceramide.

The present invention also provides a method for enhancing immunity of mammals including humans, which comprises administering effective doses of LF or its hydrolyzate and ceramide.

Brief description of the drawings

Fig. 1 is a schematic diagram of the total IgE concentration in serum after mice were sensitized with OVA antigen in the 1% bovine LF oral intake group (■: n=7) and the control group (□: n=8) (mean ±SD; *p<0.05).

Figure 2 is a graph showing OVA-specific IgE antibody levels in serum (mean ± SD; *p<0.05).

Figure 3 is a schematic representation of the IL-4 production capacity of mouse splenocytes (cells pooled in each group). ●: 1% bovine LF oral intake group, ■: control group.

Fig. 4 is to 2.2% heat-treated bovine LF oral intake group (n=19) and control group (n=17), after sensitization (3 times) with OVA antigen, the total IgE antibody concentration in mouse serum Schematic (*p<0.05).

Figure 5 is a graph showing OVA-specific IgE antibody levels in serum (*p<0.05).

Fig. 6 is a schematic diagram showing the production of OVA-specific IFN-γ by mouse splenocytes (bovine LF oral intake group: n=11, control group: n=10).

Figure 7 is a graph showing OVA-specific IL-4 production by mouse splenocytes (*p<0.05).

Fig. 8 is to 2.2% heat-treated bovine LF oral intake group (n=8) and control group (n=7), after sensitization (4 times) with OVA antigen, mouse splenocytes produce OVA-specificity Schematic representation of IFN-γ.

Fig. 9 is a schematic diagram showing the production of OVA-specific IL-4 by mouse splenocytes (control group: 7 mice, 2.2% bLF: 8 mice).

Figure 10 is a schematic diagram of the total IgE antibody concentration in mouse serum after sensitization with OVA antigen for 1% bovine LF hydrolyzate oral intake group (n=7) and control group (n=8) (*p< 0.05).

Figure 11 is a graphical representation of the effect on OVA-specific IgE antibody levels in serum.

Figure 12 is a schematic representation of OVA-specific IFN-γ production by mouse splenocytes.

Figure 13 is a schematic representation of OVA-specific IL-4 production by mouse splenocytes.

Figure 14 is a graph of IL-12 production in the culture supernatant after culturing mouse peritoneal macrophages in the presence of 100 μg/ml bovine LF (**p<0.01).

Figure 15 shows the IFN-γ in the culture supernatant when cultured mouse splenocytes after intraperitoneal administration of lactosylceramide and splenocytes without administration of lactosylceramide in the presence or absence of 100 μg/ml bovine LF Schematic diagram of yield.

Best Mode for Carrying Out the Invention

In many allergic diseases, allergen-specific IgE is known to be involved in their pathogenesis. In fact, allergen-specific IgE is frequently found in the serum of patients with allergic diseases (Allergy Clin. Immunol., 16, 161, 1996). Therefore, inhibition of IgE production is considered to be an effective method for the prevention and treatment of allergy.

A cytokine produced by CD4 ⁺ helper T cells (Th cells) plays an important role in IgE production. Depending on the cytokines produced and their functions, Th cells are roughly divided into two subgroups, Th1 cells related to cellular immunity and Th2 cells related to humoral immunity, and Th2 cells are involved in IgE production (Journal of Immunology ( J. Immunol.), 136, 2348, 1986).

Th1 cells and Th2 cells are interconnected, and the cytokines produced by them inhibit each other in differentiation and functional expression. Specifically, Th2 cells produce IL-4, IL-5, and IL-6 to control IgE production; conversely, IFN-γ produced by Th1 cells suppresses IgE production by inhibiting IgE class switching and Th cell responses. Accordingly, Th2 cell responses increase IgE production and promote allergic disease (Nature, 383, 787, 1996). Therefore, restoring Th1/Th2 balance to normal by inducing Th1 cell response can prevent and improve allergic diseases. Attempts have thus been made to screen food components for factors that induce Th1 responses. Some types of lactic acid bacteria (Int.Arch.Allergy Immunol., 115,278,1998), nucleotides (Int.Arch. Allergy Immunol.), 122, 33, 2000) and the like show this effect.

In recent years, the biodefense effects of orally administered LF in various animal models have been reported. Therefore, the present inventors first conducted a study using mice on the production of total IgE and antigen-specific IgE in serum when mice were allowed to orally ingest LF ad libitum. It was found that ad libitum oral intake of LF suppressed total IgE antibody production and antigen-specific IgE production.

It is known that the physiological activity of LF is decreased by heat treatment (J. Pediatr., 90, 29, 1977). It was therefore hypothesized that heat treatment of LF would reduce the ability of LF to suppress total and antigen-specific IgE production. A study was then performed on the possible effect of heat-treated LF on total IgE antibody production and antigen-specific IgE production. In addition, splenocytes were cultured in the presence of OVA, and the levels of cytokines (IFN-γ, IL-4) in the culture supernatant were measured. As a result, it was found that even when LF was heated, its inhibitory activity against antigen-specific IgE antibody production and IL-4 production was not lost.

It was found that total IgE antibody production and antigen-specific IgE production were significantly suppressed when LF or heat-treated LF was orally ingested compared to controls. It was also found that when splenocytes sensitized with an antigen were cultured in the presence of the antigen, the production of IL-4 was significantly decreased compared to the control group.

The possible effect of hydrolyzed LF on total IgE antibody production and antigen-specific IgE production when it was orally ingested ad libitum was then investigated. In addition, splenocytes sensitized with an antigen were cultured in the presence of the antigen, and the levels of cytokines (IFN-γ, IL-4) in the supernatant were measured. As a result, it was found that total IgE antibody production and antigen-specific IgE production tended to decrease in the LF hydrolyzate intake group. On the other hand, in the LF hydrolyzate ingestion group, it was confirmed that IFN-γ production tended to increase in the presence of OVA regardless of the concentration, but for IL-4, there was no difference between the LF hydrolyzate ingestion group and the control group. No difference was observed between.

IL-12 is a cytokine produced by antigen-presenting cells such as monocytes, macrophages and dendritic cells, which plays an important role in the production of IFN-γ and the induction of helper T cells to differentiate into Th1 (Blood ), 84, 4008, 1994; Leukoc. Biol., 55, 280, 1994). Gazzinelli et al. reported that mice were infected with Toxoplasma gondii (Toxoplasmagondii), which caused the spleen or intraperitoneal cells to increase the production of IL-12, and then administered anti-IL-12 antibodies to mice to reduce the production of IL-12 by spleen cells and make IL-12 4 and IL-10 production increased (J. Immunol., 153, 2533, 1994). In addition, Nastala et al. found that administration of IL-12 to cancer-transplanted mice inhibited cancer growth and increased blood IFN-γ concentration (J. Immunol., 153, 1697, 1994).

Therefore, IL-12 production was studied by culturing mouse peritoneal macrophages induced in thioglycolic acid medium with LF. It was found that LF induced macrophages to produce IL-12.

Atopic dermatitis, previously considered a mild form of dermatitis, is now considered an unmanageable disease that occurs at all ages (infants to adults) and has become a serious social problem. Disturbances in the metabolism of sphingolipids in the epidermis have been considered as possible etiologies. Indeed, a decrease in the amount of ceramides in the epidermis is observed in many patients with atopic dermatitis. Ceramides are also used as nutraceuticals and functional foods. The present inventors therefore hypothesized that the combined oral intake of LF and ceramide enhanced the effect of LF in preventing and improving allergic diseases over that of LF alone.

Therefore, the present inventors conducted a study on the possible effect of the combination of LF and ceramide on IFN-γ production. It was found that the combination of LF and ceramide significantly increased the production of IFN-γ in splenocytes. Among sphingolipids, sphingolipids (sphingosine, sphingomyelin, lysophosphatidylcholine) in yogurt (fermented milk) are known to promote the production of IFN-β (Biotherapy, 115-123 , 1994). It has also been reported that blood IFN-β and IFN-γ in infected mice was increased by administration of sphingosine (Osada, K. et al., Animal Cell Technology: Development towards the ^{21st Century} ), 1067-1071, 1995).

Based on these results, it is considered that the inhibitory effect of oral ingestion of LF on the production of antigen-specific IgE antibodies may be attributable to the suppression of activation of IgE-producing B lymphocytes through the inhibition of IL-4 production and also to the suppression of activation of IgE-producing B lymphocytes through the increase of IL-4. -12 produces a shift towards a Th1 response in the Th1/Th2 balance. Therefore, oral intake of IF is effective for preventing and improving allergic inflammatory diseases. Accordingly, adding an effective amount of LF as a food supplement can prevent and improve allergic diseases through diet, and LF can also be used as a drug against these diseases. Examples of allergic inflammatory diseases are chronic bronchial asthma, atopic dermatitis, hay fever (allergic rhinitis), allergic vasculitis, allergic conjunctivitis, allergic gastroenteritis, allergic liver disease, allergic bladder inflammation and allergic purpura. LF is also expected to prevent and ameliorate antibody-induced nephritis or similar diseases.

Since the combined application of LF and ceramide significantly increased the production of IFN-γ, it is understandable that the combined application of LF and ceramide inhibits the production of IgE antibodies by shifting towards Th1 dominance in the Th1/Th2 balance. IFN-γ is known to exhibit antiviral activity, antitumor activity, induce cytotoxic T lymphocyte effect, induce natural killer cell (NK cell) activity effect, activate neutrophil effect, activate macrophage effect, and promote MHC II expression Effect, promote IL-2 receptor expression effect, promote Fc receptor expression effect and so on. On the other hand, IFN is essentially a biological substance, which participates in the defense mechanism of the living body against foreign substances, has extremely high selective toxicity, and is highly safe to the living body. When food or the like containing the composition of LF and ceramide is orally ingested, the composition stimulates living cells to promote the secretion of IFN-γ and exerts the above-mentioned physiological activity of IFN-γ. Therefore, compositions containing LF and ceramide in combination can be used as antiviral, antineoplastic and/or antibacterial agents. Regarding the safety of LF, in the acute toxicity test and subacute toxicity test of bovine LF with mice, even when bovine LF was administered at the maximum dose (2,000 mg/kg body weight/day), absolutely no abnormalities were observed (milk Science (Milk Science), 48(3), 227-232, 1999). As for the safety of ceramides, the LD ₅₀ of rice ceramide (a ceramide obtained from the seeds of Oryza Sativa Linne) is 5,000 mg/kg or more for mice (FOODStyle, 4(10), 99- 105, 2000).

Examples of LF usable in the present invention may include commercially available LF, obtained from mammals (such as humans, cows, sheep, goats, horses, or colostrum, transitional milk, normal milk, late-lactating milk, etc., or skimmed milk or LF isolated from whey obtained from these milks, its hydrolyzate produced with acid or enzyme, by using hydrochloric acid apoLF obtained by removing iron with , citric acid or the like, and metal saturated or partially saturated LF obtained by chelating apoLF with metals such as iron, copper, zinc and manganese.

To prepare the LF hydrolyzate in the present invention, known hydrolysis methods can be used. When an enzyme is used for hydrolysis, enzymes other than trypsin, such as hepsin and papain, can be used, although trypsin is used in the present invention. No restriction is imposed on the enzyme as long as the hydrolyzate has the activity according to the invention. The hydrolyzate can be concentrated by inactivating the enzyme with heat followed by fractionation by methods known per se in the art, such as ultrafiltration. The resulting liquid hydrolyzate can be used directly, or after lyophilization.

Recombinant LF includes a polypeptide having an amino acid sequence substantially identical to that disclosed by Orla M. Conneely et al. (US Patent 5,766,939). Also included are naturally occurring alleles as well as modified LFs by insertion, substitution or deletion of one or more amino acids compared to native LF. Recombinant LF also includes recombinant LF expressed in transgenic animals such as cattle. Its glycosylation pattern may differ from native LF obtained from human milk.

Ceramides usable in the present invention include ceramide-related substances. For example, sphingolipids, particularly preferably glycosphingolipids. Examples of glycosphingolipids may include cerebrosides (which are the simplest glycosphingolipids found in milk, brain and kidney), sulfatides with a sulfate group added to cerebrosides, ceramide oligohexosides With several neutral sugar molecules added to cerebrosides and gangliosides with cyanuric acid added to cerebrosides. The structures of more than 100 kinds of glycosphingolipids have been determined depending on the differences in the sugar chains of glycosphingolipids.

Glycosphingolipids are included as long as they are effective in the present invention. Preferable examples may include lactosylceramide, galactosylceramide and glycosylceramide. In addition, sphingomyelin (contained in milk), which is a type of phospholipid, is also preferred. In addition, asymmetric synthesis technology or the like has made it possible to chemically synthesize natural ceramides, and optically active ceramides are being developed. They include ceramide 2 and ceramide 3 (Cosmo Farm Co., Ltd.). These ceramides can be used in the present invention as long as they are effective in the present invention. Moreover, plant-derived ceramides are attracting attention and developing applications. Among rice-derived sphingolipids, for example, a hydrophobic ceramide with a fatty acid bonded to a long-chain basic sphingosine through an acid-amide structure is the basic backbone of similar animal sphingolipids. Rice-derived sphingolipids are diverse in molecular species depending on differences in the number of carbon atoms of long-chain basic sphingosine and fatty acids and the presence or absence of hydroxyl groups or double bonds. According to a report by Professor Fujino of Obihiro University of Agriculture and Veterinary Medicine, there are at least more than 20 molecular species of sphingolipids. The present invention includes these ceramides.

Regarding the content of LF (including all LF, LF derivatives having physiological activity comparable to LF, and hydrolyzed products of LF and LF derivatives) and ceramide in the present invention, it is estimated that the ceramide content ranges preferably from 0.0001 to 0.1 wt % , more preferably from 0.0005 to 0.01 wt%, particularly preferably from 0.0001 to 0.05 wt%, all wt% are based on the total weight of the composition, and the estimated content of LF is preferably from 0.005 to 10 wt%, particularly preferably from 0.01 to 5 wt% , most preferably from 0.01 to 3 wt%, all wt% based on the total weight of the composition. However, it is believed that those skilled in the art can easily determine the optimum content in each target composition, for example, by the following experiments. Incidentally, the amount of ceramide in milk is known and is believed to serve, at least to some extent, as a guide for the amount of ceramide to be added.

The present invention has demonstrated that oral intake of LF inhibits the production of antigen-specific IgE antibodies. Therefore, those skilled in the art can select the preferred form of LF applicable to the present invention from the above-mentioned various LFs and their effective amounts based on the present invention, for example, by using IgE antibody production as an indicator; depending on clinical data, clinical symptoms, etc. and patients The relationship between serum IgE to determine the dosage form and effective dose of LF for use in food, supplements, food for the sick, infant formula, health food, food for health requirements, functional food, specific health food, drug or etc. Accordingly, the types of LF determined as described above and their effective doses are included in the present invention. The incorporation or processing of LF into foodstuffs and the like is well known and commonly used. When an effective amount of LF is used as a drug, it can be used in various formulations known to those skilled in the art.

Example

The present invention will hereinafter be described based on tests and examples. But it should be kept in mind that the invention is not limited to these tests and examples.

In the following experiments, 3-week-old male immature BALB/c mice (Japan SLC, Inc., Shizuoka, Japan) were used in tests 1-4 and 6, and 8-week-old female BALB/c mice Rats (Japan SLC, Inc., Shizuoka, Japan) were used in Test 5. The significance test between each control group and its corresponding test group was carried out by Student's t test.

To test the activity of 1 bovine LF (bLF) to inhibit the production of IgE antibody

During the test period, the mice (n=7) in the LF-administered group were allowed to ingest a feed containing casein as a protein source (based on AIN76), and a diet containing 1% bovine LF (bLF, DMV JapanK.K. product) of water. Mice (n=8) in the control group were given the above-mentioned feed and bLF-free water ad libitum.

On days 5 and 19 after the start of the test, each mouse was intraperitoneally immunized with 10 µg of ovalbumin (OVA, product of SEIKAGAKU CORPORATION) and 4 mg of aluminum hydroxide. On day 26, blood was taken from the orbit, and the total IgE antibody level and the OVA-specific IgE antibody level in the serum were measured by ELISA. Both total IgE antibody levels (Fig. 1) and OVA-specific IgE antibody levels (Fig. 2) in serum of mice in the bLF group were lower than those in the control group (*p<0.05).

Test 2 bLF activity to inhibit IL-4 production

In Test 1, the spleen was excised to prepare a splenocyte suspension. After hemolysis, splenocytes were cultured at various concentrations (5, 10, 50 and 100 μg/mL) in the presence of OVA for 72 hours. After culturing, the IL-4 level in each culture supernatant was measured by ELISA. In the bLF-administered group (n=7), the IL-4 levels at each OVA concentration were lower than those in the control group (n=8).

Test 3 The activity of heat-treated bLF in inhibiting the production of IgE antibodies and cytokines

A feed [bLF(+)] obtained by adding 2.2% bLF to the feed of Test 1 was heated at 70°C for 1 hour. As a control, a feed to which no bLF was added [bLF(-)] was similarly heated at 70° C. for 1 hour.

During the test period, mice were allowed to ingest bLF(+) (test group) or bLF(-) (control group) ad libitum. On the 5th day, the 14th day, the 23rd day and the 33rd day after the start of the test, each mouse was treated with ovalbumin (OVA, a product of SEIKAGAKU CORPORATION) (10 μg) and aluminum hydroxide (4 mg) for peritoneal internal immunity. On the eighth day (day 30) (7 weeks old) after the third peritoneal immunization, blood was collected from the tail, and the antibody in the serum was measured by ELISA. In addition, on the 11th day (day 33) after the third peritoneal immunization and the 8th day (day 40) after the fourth peritoneal immunization, splenocytes were collected separately depending on each mouse. Splenocytes were treated with different concentrations (0, 50, 100, 200 and 400 μg/mL) of OVA in 10% FCS, 100 U/mL penicillin G, 100 μg/mL streptomycin and 5×10 ^-5 M 2-mercapto Cultured in ethanol-based RPMI1640 medium for 72 hours. After culturing, the IFN-γ level and IL-4 level in each culture supernatant were measured by ELISA.

When immunizations were performed three times, the total IgE antibody level and the OVA-specific IgE antibody level in the serum of the test group were significantly reduced compared with the corresponding results in the control group (Fig. 4 and Fig. 5). For IFN-γ production by splenocytes at each OVA concentration (0, 50, 100 and 200 μg/mL), no substantial difference was observed between the test and control groups ( FIG. 6 ). On the other hand, the production of IL-4 by splenocytes in the test group was generally lower than that of the control group at each OVA concentration, and under the stimulation of 200 μg/mL OVA, the production of IL-4 in the test group decreased more significantly than that of the control group (Figure 7).

When 4 immunizations were performed, IFN-γ production by splenocytes at various OVA concentrations (0, 50, 100, 200 and 400 μg/mL) did not produce substantial differences between groups as in the example of 3 immunizations (Figure 8). On the other hand, the production of IL-4 in the test group was generally more significantly decreased than that in the control group at each OVA concentration ( FIG. 9 ).

From the above results, it can be seen that the activity of bLF to inhibit the production of antigen-specific IgE antibody and the activity of inhibiting the production of IL-4 were not lost with heating.

Test 4 activity of hydrolyzed bLF to inhibit IgE antibody and cytokine production

(1) Preparation of hydrolyzed bLF

Trypsin ("207-09891", product of Wako Pure Chemical Industries, Ltd., for biochemical use, prepared from porcine spleen, 4,500 USP trypsin units/mg) was dissolved in sterilized PBS as a trypsin stock solution (×500, 50 mg/mL). bLF ("127-04122", product of Wako Pure Chemical Industries, Ltd., lot. KSG7724) (10 g) was dissolved in 25 mM CaCl ₂ -0.1 M Tris-HCl (pH 8.2) to form a 1% solution. After heating 1 L of bLF solution to 37°C, trypsin stock solution (2 mL) was added, followed by reaction at 37°C for 4 hours. After the reaction, the enzyme was inactivated by heating at 80° C. for 30 minutes. After centrifugation at 1,700×g for 20 minutes, the supernatant was collected and sterilized by filtration with a 0.45 μm filter, and the sample was treated with “Micro Acilyezer S1” (an electrodialysis machine, manufactured by Asahi Chemical Industry Co., Ltd.), and from the sample Remove the salt. After re-sterilisation by filtration with a 0.45 μm filter, the samples were stored at -20°C until used for testing. The yield of bLF hydrolyzate was essentially 100%. The concentration of bLF hydrolyzate was 1%.

Mice were allowed to ingest the feed of Test 1 and the solution containing 1% bLF hydrolyzate ad libitum. The control group was given the same solid feed and bLF-free water ad libitum. On the 5th and 19th days after the start of the test, each mouse was intraperitoneally immunized with ovalbumin (OVA, product of SEIKAGAKU CORPORATION) (10 µg) and aluminum hydroxide (4 mg). On the 8th day (day 26) after the second immunization, blood was collected, and the antibody in the serum was measured by ELISA. At the same time, splenocytes were collected separately depending on each mouse. Splenocytes were co-cultured with OVA (0-400 μg/mL) for 72 hours, and the levels of IFN-γ and IL-4 in each culture supernatant were measured by ELISA.

It was observed that the total IgE antibody level and the OVA-specific IgE level in the serum of the test group became lower compared with the corresponding levels in the control group ( FIGS. 10 and 11 ).

It was noted that the overall tendency of splenocytes to produce IFN-γ at different OVA concentrations was higher in the test group than in the control group ( FIG. 12 ). On the other hand, IL-4 production showed no substantial difference between the test group and the control group (Fig. 13).

Test the effect of 5 LF on the production of IL-12

Although IL-12 is a relatively recently discovered cytokine, it is known to act on T cells and NK cells to induce IFN-γ production (J. Exp. Med., 173, 869-879 , 1991; Journal of Experimental Medicine (J. Exp. Med.), 177, 1199-1204, 1993). Stimulation of cellular components such as LPS is effective for inducing IL-12 production, and IL-12-producing cells include various cells such as macrophages, B cells, and neutrophils.

The present inventors cultured peritoneal macrophages in a medium added with bLF (product of Wako Pure Chemical Industries, Ltd.), and measured the level of IL-12 in the culture supernatant by ELISA.

To mice fed diet "MF" (product of Oriental Yeast Co., Ltd) ad libitum, thioglycolic acid medium (product of DIFCO Laboratories) (2.5 mL) was each intraperitoneally injected to activate peritoneal macrophages. Four days later, Dulbeccos' phosphate buffer solution containing 1% FCS ("Dulbeccos'PBS", product of NISSUI PHARMACEUTICALCO., Ltd.) was injected into the peritoneal cavity to collect peritoneal cells. After the cells were washed twice, they were resuspended in "Dulbeccos'PBS", and after adding to a 96-well plate (2×10 ⁵ cells/0.2 mL well), the plate was incubated in a CO 2 incubator for 2 hours. After removing the non-adherent cells, the solution was treated with 10% FCS, 100 U/mL penicillin G, 100 μg/mL streptomycin, 5×10 ⁻⁵ M 2-mercaptoethanol and 100 μg/mL bLF (Wako Pure Chemical Industries, Ltd. Product) RPMI 1640 medium (200 μL) filled each well. As a control, the above medium without bLF was used. Plates were incubated for 18 hours in a CO2 incubator. After incubation, the IL-12 level in each supernatant was measured by ELISA. The result is shown in Figure 14. The addition of bLF to the medium significantly increased the production of IL-12 by macrophages.

From the above results, it can be seen that lactoferrin induces macrophages to produce IL-12.

Test the effect of 6 ceramides on the production of IFN-γ

BALB/c mice (3 weeks old, male) (Japan SLC, Inc.) were used for the test. The mice were divided into a lactosylceramide-administered group (test group) and a lactosylceramide-nonadministered group (control group) (3 mice per group) so that the mean and deviation of the body weights of the two groups of mice were substantially equal.

Used as the ceramide was lactosylceramide ("Biochemical Reagent 126-04491", product of Wako Pure Chemical Industries, Ltd., derived from milk, lotELK6191). Lactosylceramide was dispersed at a concentration of 200 μg/mL in a 0.9% NaCl solution containing 0.5% Tween20, and the resulting dispersion was stored at -20°C until use. At the time of use, the dispersion was thawed, and an equal amount of sterile PBS was added thereto. After thorough mixing, a lactosylceramide solution (200 μL per mouse) was intraperitoneally administered to the mice every day for 14 days, except Sundays and Saturdays (20 μg per mouse). For the control group, only the solvent (0.9% NaCl solution containing 0.5% Tween20 and an equal amount of sterilized PBS added) was applied.

Splenocytes obtained from each mouse on the 14th day after administration were inoculated into a 96-well microculture plate ("No. 3072", manufactured by Becton Dickinson and Company) at an inoculum amount of 4 × 10 ⁵ cells per well . The culture medium used was RPMI 1640 medium (“No. 11875-093”, product of Gibco BRL), containing 10% fetal bovine serum (product of Japan Biotest Laboratory, lot 10086-1), 100 U/mL penicillin, 100 μg /mL streptomycin and 5×10 ^-5 M2-mercaptoethanol. The culture was carried out for 4 rounds, and the splenocytes were cultured for 20 hours in the presence of bLF (product of Meggle GmbH, 0 or 100 μg/mL). After the culture, IFN-γ in each culture supernatant was measured with an ELISA kit (product of ENDOGEN Inc.). Data were analyzed with two-way categorical ANOVA (2x2, significance level: 5%) taking lactosylceramide administration and bLF stimulation as factors.

The result is shown in Figure 15. When ceramide was not administered, no IFN-γ production was observed regardless of the presence or absence of bLF. When ceramide was pre-administered, a significant increase in IFN-γ production was observed both in the absence and presence of bLF. Since an interaction was observed between these two factors, ceramide and bLF (p=0.0016), administration of lactosylceramide was found to increase bovine LF-stimulated IFN-γ production.

Example 1

Bovine LF was added at a concentration of 0.01% to "EPITOLESS" (a product of MEIJI MILK PRODUCTSCO., LTD), which is milk for milk allergy patients, which does not use whey protein as a raw material, and has the following formula. Element per 100g product Protein (N×6.38) g 14.5 Fat g 20.0 hydrocarbon g 60.0 (lactose) g (Sucrose 8.0) (soluble polysaccharide) g (52.0) Ash g 2.5 water g 3.0 energy kcal 478 Vitamin A IU 2,000 Vitamin B1 mg 0.6 Vitamin B2 mg 0.9 Vitamin B6 mg 0.3 Vitamin B12 µg 4 Vitamin C mg 50 Vitamin D IU 370 Vitamin E (alpha-tocopherol) mg 6 Vitamin K µg 25 pantothenic acid mg 2 niacin mg 6 folic acid mg 0.2 β-carotene µg Inositol mg 20 Linoleic acid g 1.7 alpha-linolenic acid g 0.34 taurine mg 27 calcium mg 400 magnesium mg 42 Potassium mg 525 sodium mg 150 phosphorus mg 230 chlorine mg 320 iron mg 6.5 copper µg 320 zinc mg 2.8

Example 2

Bovine LF was added at a concentration of 0.01% to hydrolyzed milk "MEIJI NOBIYAKA" a product of MEIJIMILK PRODUCTS CO., LTD), which contains whey protein as a raw material. Element per 100g product Protein (N×6.38) g 12.2 Fat g 25.0 hydrocarbon g 57.5 (lactose) g (29.5) (soluble polysaccharide) g (28.0) Ash g 2.2 water g 3.0 energy kcal 504 Vitamin A IU 2,000 Vitamin B1 mg 0.6 Vitamin B2 mg 0.9 Vitamin B6 mg 0.3 Vitamin B12 µg 4 Vitamin C mg 50 Vitamin D IU 375 Vitamin E (alpha-tocopherol) mg 6 Vitamin K µg 25 pantothenic acid mg 2 niacin mg 6 folic acid mg 0.2 β-carotene µg 70 Inositol mg 25 Linoleic acid g 2.6 alpha-linolenic acid g 0.66 taurine mg 27 calcium mg 360 magnesium mg 40 Potassium mg 510 sodium mg 170 phosphorus mg 200 chlorine mg 300 iron mg 6.5 copper µg 320 zinc mg 2.8

Industrial Applicability

It was found by the present invention that oral intake of LF suppresses the production of IgE antibodies in vivo.

Claims (26)

CLAIMS 1. A mammalian (including human) antigen-specific IgE antibody production inhibitor comprising lactoferrin or its hydrolyzate as an active ingredient. 2. An inhibitor according to claim 1, further comprising ceramide as an active ingredient. 3. A composition for controlling immunity by suppressing the production of antigen-specific IgE antibodies in mammals (including humans), comprising lactoferrin or its hydrolyzate as an active ingredient. 4. A composition according to claim 3, further comprising ceramide as an active ingredient. 5. An IFN-γ production enhancer composition for mammals (including humans), comprising lactoferrin or its hydrolyzate and ceramide as active ingredients. 6. An antiviral agent composition for mammals including humans, comprising lactoferrin or its hydrolyzate and ceramide as active ingredients. 7. An antibacterial agent composition for mammals including humans, comprising lactoferrin or its hydrolyzate and ceramide as active ingredients. 8. An antitumor agent composition for mammals including humans, comprising lactoferrin or its hydrolyzate and ceramide as active ingredients. 9. An immunopotentiator composition for mammals including humans, comprising lactoferrin or its hydrolyzate and ceramide as active ingredients. 10. Use of lactoferrin or its hydrolyzate for the preparation of an inhibitor of antigen-specific IgE antibody production in mammals (including humans). 11. Use according to claim 10, further adding ceramide. 12. Use of lactoferrin or its hydrolyzate for preparing a composition for controlling immunity by inhibiting the production of antigen-specific IgE antibodies in mammals (including humans). 13. Use according to claim 12, further adding ceramide. 14. Use of lactoferrin or its hydrolyzate and ceramide for the preparation of an IFN-γ production enhancer composition for mammals including humans. 15. Use of lactoferrin or its hydrolyzate and ceramide for the preparation of an antiviral agent composition for mammals including humans. 16. Use of lactoferrin or its hydrolyzate and ceramide for the preparation of an antibacterial agent composition for mammals including humans. 17. Use of lactoferrin or its hydrolyzate and ceramide for the preparation of an antitumor agent composition for mammals including humans. 18. Use of lactoferrin or its hydrolyzate and ceramide for preparing an immunopotentiator composition for mammals including humans. 19. A method for inhibiting IgE antibody production in mammals including humans, comprising administering an effective amount of lactoferrin or a hydrolyzate thereof. 20. A method of inhibiting IgE antibody production in mammals including humans, comprising administering effective amounts of lactoferrin or a hydrolyzate thereof and ceramide. 21. A method for treating viral diseases in mammals including humans, comprising administering effective amounts of lactoferrin or its hydrolyzate and ceramide. 22. A method of treating bacterial infection in mammals including humans, comprising administering an effective amount of lactoferrin or its hydrolyzate and ceramide. 23. A method for treating tumors in mammals including humans, comprising administering effective amounts of lactoferrin or its hydrolyzate and ceramide. 24. A method of modulating IgE antibody production in mammals including humans, comprising administering an effective amount of lactoferrin or a hydrolyzate thereof. 25. A method of increasing IFN-γ production in mammals including humans, comprising administering effective amounts of lactoferrin or its hydrolyzate and ceramide. 26. A method for enhancing immunity of mammals including humans, comprising administering effective amounts of lactoferrin or its hydrolyzate and ceramide.

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