CN1205998C - 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

Antigenic specificity IgE antibody produces inhibitor

Technical field

The present invention relates to IgE and produce inhibitor, it effectively prevents or improves allergic disease such as atopic dermatitis.The invention still further relates to stimulates the compositions of live body immune system with enhance immunity.

Background technology

Lactoferrin (LF) be a kind of ferrum in conjunction with glycoprotein, it does not exist only in the Ruzhong, also is present in the exocrine secretion, as saliva, tear, bile and pancreatic juice.It can also be discharged by the neutrophilic leukocyte that is activated at inflammation part.

LF has the broad-spectrum biological activity.For example comprise antimicrobial acivity, antiviral activity, anti-tumor activity, inhibition cancerometastasis activity and so on or the like.

In addition, LF influences cytokine secretion.For example LF promotes human neutrophilic leukocyte secreting leukocytes mesonium 8 (IL-8), control IL-1, the secretion of IL-6 and TNF α, and the expression of inhibition GM-CSF gene level.

Selectivity produces cytokine and play protective effect or nosetiology effect in various diseases.For example, most of allergy are I allergic reaction types, and it is associated with IgE antibody.It is inductive by the process that relates to IL-4 that this IgE produces, on the other hand, this IgE produce be subjected to relating to interferon- _γ(IFN- _γ) process control.IFN- _γGeneration be inductive by its upper IL-12 and IL-18.

IFN- _γAlso demonstrate antiviral activity, antimicrobial acivity and anti-tumor activity, these activity depend on various immunoloregulation functions (Journal of Immunology (J.Immunol.), 130,2011-2013,1983; Institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 85,4874-4878,1988).

Reports such as Nakajima, when behind the oral cattle LF (bLF) mouse boosting cell being cultivated in the presence of mitogen (Con A), IFN- _γGeneration increase, but the generation of IL-4 does not increase (biomedical research (Biomedical Research), 29 (1), 27-33,1999).

As mentioned above, LF participates in multiple biological activity and production of cytokines and regulation and control.Therefore target of the present invention is to disclose the new biological activity of LF, and based on this activity, provides and contain the medicine and the functional food of LF as active component.Another target of the present invention is a material of finding that but a kind of per os is taken in, when its during with the LF associating, have the bioactive effect of increase LF.

Of the present invention open

The inventor finds, when take in LF with the immune per os of a kind of main food allergens ovalbumin (OVA), during through the mice of heat treated LF or LF hydrolyzate, the generation of OVA-specific IgE antibody is suppressed, and the generation of the antigenic specificity IL-4 in the spleen cell cultures system also is suppressed.They find that also when cultivating macrophage in the presence of LF, macrophage produces IL-12 to be increased.In addition, they also find, unite and use LF and ceramide, significantly increase IFN-in the spleen cell cultures system _γGeneration.Think and take in LF by per os to suppress antigenic specificity IgE production of antibodies be by suppressing the activation that IL-4 produces the bone-marrow-derived lymphocyte that suppresses to produce IgE antibody, and move to the Th1 advantage in the Th1/Th2 balance by the generation of increase IL-12 and to cause.That is, per os is taken in LF can prevent the reduction of moving the cellular immunization that takes place by replying to Th2, and can prevent or improve the nephritis that is caused by allergic disease or antibody response.The combination of LF and ceramide significantly increases IFN- _γThe generation combination effectively enantiopathy viral disease, bacterial infection or the tumor that show LF and ceramide.

Specifically, the invention provides a kind of mammal (comprising the people) IgE antibody and produce inhibitor, it comprises active component LF or its hydrolyzate.

The present invention also provides a kind of and produces the compositions of controlling immunity by suppressing mammal (comprising the people) IgE antibody, and it comprises active component LF or its hydrolyzate.

The present invention also is provided for comprising people's mammiferous IFN- _γProduce enhancer composition, antiviral agent compositions, antimicrobial composition or antitumor agent compositions, it comprises LF or its hydrolyzate and ceramide.

The present invention also is provided for comprising people's mammiferous immunopotentiator composition, and it comprises LF or its hydrolyzate and ceramide.

The present invention also provides LF or its hydrolyzate to be used to prepare the purposes that mammal (comprising the people) IgE antibody produces inhibitor.

The present invention also provides LF or its hydrolyzate to be used for preparation by suppressing the purposes that mammal (comprising the people) IgE antibody produces the compositions of controlling immunity.

The present invention also provides LF or its hydrolyzate and ceramide to be used to prepare the mammiferous IFN-that is used to comprise the people _γProduce the purposes of enhancer composition, antiviral agent compositions, antimicrobial composition or antitumor agent compositions.

The present invention also provides LF or its hydrolyzate and ceramide to be used to prepare to be used to the purposes of the mammiferous immunopotentiator composition that comprises the people in addition.

The present invention also is provided at and suppresses the method that IgE antibody produces in the mammal that comprises the people, and it comprises LF or its hydrolyzate of using effective dose.

The present invention also is provided at and regulates the method that IgE antibody produces in the mammal that comprises the people, and it comprises LF or its hydrolyzate of using effective dose.

The present invention also is provided in the mammal that comprises the people increases IFN- _γThe method that produces, it comprises LF or its hydrolyzate and the ceramide of using effective dose.

The present invention also provides the method that strengthens the mammalian immune that comprises the people, and it comprises LF or its hydrolyzate and the ceramide of using effective dose.

Brief Description Of Drawings

Fig. 1 be 1% N of LF per os absorption group (■: n=7) and matched group (: n=8) with mice with the sensitization of OVA antigen after the sketch map (meansigma methods ± SD of total IgE concentration in the serum; * p＜0.05).

Fig. 2 is the sketch map (meansigma methods ± SD of OVA-specific IgE antibody level in the serum; * p＜0.05).

Fig. 3 is the sketch map that mouse boosting cell (in each group cell being merged) produces the IL-4 ability.●: 1% N of LF per os absorption group, ■: matched group.

Fig. 4 is to 2.2% through heat treated cattle LF per os absorption group (n=19) and matched group (n=17), with OVA antigen sensitization (3 times) afterwards, and the sketch map (* p＜0.05) of total IgE antibody concentration in the mice serum.

Fig. 5 is the sketch map (* p＜0.05) of OVA-specific IgE antibody level in the serum.

Fig. 6 is mouse boosting cell (cattle LF per os absorption group: n=11, a matched group: n=10) produce OVA-specificity IFN- _γSketch map.

Fig. 7 is the sketch map (* p＜0.05) that mouse boosting cell produces OVA-specificity IL-4.

Fig. 8 is to 2.2% through heat treated cattle LF per os absorption group (n=8) and matched group (n=7), and with OVA antigen sensitization (4 times) afterwards, mouse boosting cell produces OVA-specificity IFN- _γSketch map.

Fig. 9 is the sketch map that mouse boosting cell (matched group: 7 mices, 2.2%bLF:8 mice) produces OVA-specificity IL-4.

Figure 10 is to 1% N of LF hydrolyzate per os absorption group (n=7) and matched group (n=8), after the sensitization of OVA antigen, and the sketch map (* p＜0.05) of total IgE antibody concentration in the mice serum.

Figure 11 is the sketch map to the effect of OVA-specific IgE antibody level in the serum.

Figure 12 is that mouse boosting cell produces OVA-specificity IFN- _γSketch map.

Figure 13 is the sketch map that mouse boosting cell produces OVA-specificity IL-4.

Figure 14 cultivates after the mouse peritoneum macrophage sketch map of IL-12 output in culture supernatant (* * p＜0.01) in the presence of 100 μ g/ml cattle LF.

Figure 15 is under 100 μ g/ml cattle LF existence or non-existent condition, when the cultivation peritoneum is used the mouse boosting cell behind the lactoside ceramide and is not used the splenocyte of lactoside ceramide, and IFN-in the culture supernatant _γThe sketch map of output.

Implement optimal mode of the present invention

In many allergic diseases, known allergen specific IgE participates in its morbidity.In fact, in allergic disease patient's serum, often find allergen-specific IgE (allergy clinical immunology (Allergy Clin.Immunol.), 16,161,1996).Therefore think that suppressing the IgE generation is to prevent and treat allergic effective ways.

In IgE produced, a kind of cytokine that is produced by CD4 ' helper T cell (Th cell) played an important role.Depend on the cytokine and the function thereof that are produced, the Th cell roughly is divided into two subgroups, Th1 cell relevant and the Th2 cell relevant with humoral immunization with cellular immunization, what participate in the IgE generation is Th2 cell (Journal of Immunology (J.Immunol.), 136,2348,1986).

Th1 cell and Th2 cell are interrelated, and in differentiation and functional expression, the cytokine by its generation suppresses each other.Specifically, Th2 cell generation IL-4, IL-5 and IL-6 produce with control IgE; On the contrary, the IFN-of Th1 cell generation _γSuppress the IgE generation by suppressing conversion of IgE classification and Th cell response.Correspondingly, the Th2 cell response increases the IgE generation and promotes allergic disease (nature (Nature), 383,787,1996).Therefore can prevent and improve allergic disease by inducing the Th1 cell response that the Th1/Th2 balance is returned to normally.So once attempted the factor that screening induces Th1 to reply in composition of food.Found lactic acid bacteria (the international allergy immunology document (Int.Arch.Allergy Immunol.) of some type before this, 115,278,1998), nucleotide (international allergy immunology document (Int.Arch.Allergy Immunol.), 122,33,2000) and so on or the like demonstrate this effect.

In recent years, reported the biophylaxis effect of oral LF in various animal models.Therefore the inventor has at first carried out a research with mice, and content is about when allowing the random per os of mice take in LF, the generation of IgE and antigenic specificity IgE always in the serum.Found that random per os absorption LF suppresses the generation of total IgE production of antibodies and antigenic specificity IgE.

The physiologically active of known LF is through heat treatment and descend (pediatrics magazine (J.Pediatr.), 90,29,1977).Therefore hypothesis is heat-treated LF can suppress the ability that total IgE produces and antigenic specificity IgE produces by reduction LF.So carried out about through heat treated LF total IgE antibody being produced and the possible Study on Effect of antigenic specificity IgE generation.In addition, in the presence of OVA, cultivate splenocyte, and measure cytokine (IFN-in the culture supernatant _γ, IL-4) level.Even found that LF through heating, it is not lost the inhibition activity that the antigen specific IgE antibody produces and IL-4 produces yet.

Have been found that as LF or when heat treated LF per os is taken in compare with matched group, the generation of total IgE production of antibodies and antigenic specificity IgE is subjected to remarkable inhibition.Also find to compare with matched group in the time will cultivating in the presence of this is antigenic with the splenocyte of antigen sensitization, the generation of IL-4 significantly reduces.

Studied then when it produces total IgE antibody and possible effect of antigenic specificity IgE generation when the random per os of the LF of hydrolysis is taken in.In addition, will in the presence of this is antigenic, cultivate with the splenocyte of antigen sensitization, and the cytokine (IFN-in the mensuration supernatant _γ, IL-4) level.Found that in LF hydrolyzate absorption group total IgE antibody produces and antigenic specificity IgE generation has the tendency of reduction.On the other hand, in LF hydrolyzate absorption group, confirm IFN-in the presence of the OVA that does not consider concentration _γGeneration the trend of increase is arranged, but, between LF hydrolyzate absorption group and matched group, do not observe difference for IL-4.

IL-12 is a kind of cytokine that is produced by antigen-presenting cell such as mononuclear cell, macrophage and dendritic cell, and it is at IFN- _γGeneration and induce helper T cell to be divided among the Th1 to play an important role (blood (Blood), 84,4008,1994; Leukocyte biology (Leukoc.Biol.), 55,280,1994).Report such as Gazzinelli toxoplasma gondii (Toxoplasmagondii) infecting mouse, cause that spleen or intraperitoneal cell produce IL-12 and increase, make splenocyte produce the IL-12 minimizing and use anti-IL-12 antibody to mice subsequently, and make IL-4 and IL-10 produce increase (Journal of Immunology (J.Immunol.), 153,2533,1994).In addition, discoveries such as Nastala are transplanted mice to cancer and are used the growth that IL-12 suppresses cancer, and increase IFN-in the blood _γConcentration (Journal of Immunology (J.Immunol.), 153,1697,1994).

So, by will be in thioglycollate medium inductive mouse peritoneum macrophage cultivate the generation of research IL-12 with LF.Found that LF induces macrophage to produce IL-12.

Thought before that atopic dermatitis was a kind of dermatitis of gentle form, but thought that now it is all generable restive disease of a kind of institute has age (baby is to the adult), and become serious social concern.The metabolism disorder of sphingolipid is considered to the possible cause of disease in epidermis.In fact, in many atopic dermatitis patients, observe the minimizing of ceramide amount in the epidermis.Ceramide is also as nutriment and functional food.Therefore the inventor draws hypothesis, and the associating per os is taken in LF and ceramide, has strengthened LF in prevention with improve effect in the allergic disease, surpasses and uses LF separately.

Therefore, the inventor has carried out making up IFN-about LF and ceramide _γThe possible Study on Effect that produces.Found that LF and ceramide combination significantly having increased IFN-in the splenocyte _γGeneration.Among sphingolipid, known sphingolipid (sphingol, sphingomyelins, LYSO-PHOSPHATIDYLCHOLINE LYSOPC) in yogurt (milk of fermentation) promotes the generation (biotherapy (Biotherapy), 115-123,1994) of IFN-β.Also reported by using sphingol and increased infecting mouse blood IFN-β and IFN- _γ(Osada, K. etc., zooblast technology: to 21 ^StThe development in century (Animal Cell Technology:Development towards the 21 ^StCentury), 1067-1071,1995).

According to these results, think that per os absorption LF is attributable to produce the activation that has suppressed to produce the bone-marrow-derived lymphocyte of IgE by suppressing IL-4 to the inhibitory action of antigen specific IgE antibody generation, also is attributable to by increase IL-12 generation in the Th1/Th2 balance to moving that Th1 replys.Therefore, per os absorption IF is effective to preventing and improving allergia inflammatory disease.Correspondingly, the LF that adds effective dose can and improve allergic disease by the diet prevention as replenishing of food, and LF also can be used as the medicine at these diseases.The example of allergia inflammatory disease is chronic bronchial asthma, atopic dermatitis, pollinosis (allergic rhinitis), allergic vasculitis, anaphylaxis conjunctivitis, allergic gastroenteritis, anaphylaxis hepatopathy, anaphylaxis cystitis and anaphylactoid purpura.Estimate that LF also can prevent and improve nephritis or the similar disease that antibody causes.

Because use in conjunction LF and ceramide significantly increase IFN- _γGeneration, be appreciated that use in conjunction LF and ceramide are by suppressing the IgE production of antibodies to Th1 advantage migration in the Th1/Th2 balance.Known IFN- _γShow antiviral activity, anti-tumor activity, inducing cytotoxic T lymphocyte effect, induce natural killer cell (NK cell) active effect, activation neutrophilic leukocyte effect, activated macrophage effect, promote MHC II expression effect, promote IL-2 expression of receptor effect, promote Fc expression of receptor effect and so on or the like.On the other hand, IFN comes down to a kind of biological substance, and the defense mechanism that it participates in live body opposing foreign substance has high selection toxicity, and to the live body high safety.When the per os such as food that contain LF and ceramide compositions were taken in, said composition stimulated active somatic cell to promote IFN- _γSecretion and bring into play above-mentioned IFN- _γPhysiologically active.Therefore, unite the compositions that contains LF and ceramide and can be used as antiviral agent, antitumor agent and/or antibacterial.About the safety of LF, in the acute toxicity test and subacute toxicity test of the cattle LF that carries out with mice, even with maximal dose (2,000 milligram/kg body weight/sky) use cattle LF, definitely do not observe unusual (newborn science (Milk Science), 48 (3) yet, 227-232,1999).As for the safety of ceramide, the LD of rice ceramide (a kind of ceramide that obtains from the seed of Oryza Sativa Linne) ₅₀To mice is 5,000 milligrams/kilogram or above (food species (FOODStyle, 4 (10), 99-105,2000).

The example that can be used for LF of the present invention can comprise commercially available LF, with the known method in this area itself (for example, ion-exchange chromatography and so on or the like) from mammal (people for example, milch cow, sheep, goat, horse or and so on animal) colostrum, transitional milk, normal breast, suckling breast in late period and so on or the like or skimmed milk or from the milk surum that these breasts obtain isolating LF, the hydrolyzate that it produces with acid or enzyme, by using hydrochloric acid, citric acid or and so on acid remove ferrum and the apoLF that obtains and by with metal (as ferrum, copper, zinc and manganese) the metal LF saturated or fractional saturation of chelating apoLF gained.

Be the LF hydrolyzate among preparation the present invention, available known method for hydrolysis.When enzyme is used for hydrolysis, available enzyme except that trypsin, for example hepsin and papain are although usefulness is trypsin in the present invention.Enzyme is not applied restriction, as long as described hydrolyzate has according to activity of the present invention.By under heating, making enzyme deactivation, use this area itself known method (as ultrafiltration) to carry out fractionated then, hydrolyzate can be concentrated.The gained liquid hydrolysate can directly be used, or uses after lyophilizing.

Reorganization LF comprises the polypeptide that has with the essentially identical aminoacid sequence of disclosed sequence (United States Patent (USP) 5,766,939) such as Orla M.Conneely.Also comprise naturally occurring allele and compare, the modified LF that forms by one or more amino acid whose insertions, displacement or disappearance with natural LF.Reorganization LF also is included in the reorganization LF that expresses in the transgenic animal (for example cattle).Its glycosylation mode can be different with the natural LF that obtains from human milk.

Can be used for ceramide of the present invention and comprise the ceramide related substances.For example, sphingolipid, preferred especially glycosyl sphingolipid.The example of glycosyl sphingolipid can comprise that cerebroside (it is the simplest glycosyl sphingolipid of finding), sulfatide have a sulfate group and add to cerebroside, the few hexoside of ceramide and have several neutral sugar molecules and add to cerebroside and ganglioside and have cyanuric acid and add to cerebroside in breast, brain and kidney.The difference that depends on the glycosyl sphingolipid sugar chain has determined to surpass the structure of 100 kinds of glycosyl sphingolipids.

Glycosyl sphingolipid is all included, as long as they are effective in the present invention.Preferred example can comprise lactoside ceramide, galactosyl ceramide and glycosyl ceramide.In addition, also preferred sphingomyelins (Ruzhong is contained), it is a kind of phospholipid.In addition, the asymmetric synthesis technology or and so on technology make the natural ceramide of chemosynthesis become possibility, and developing and have optically active ceramide.They comprise ceramide 2 and ceramide 3 (Cosmo Farm Co., Ltd.).These ceramides can be used among the present invention, as long as they are effective in the present invention.And the ceramide of plant derivation just receives publicity, and Application and Development.In the deutero-sphingolipid of rice, for example a kind of to have the hydrophobic ceramide that is bonded to the fatty acid of long-chain alkalescence sphingol by acid-amide structure be the basic framework of similar animal sphingolipid.The deutero-sphingolipid of rice has multiformity on molecular species, it depends on the existence of the difference of carbon number of long-chain alkalescence sphingol and fatty acid and oh group or two keys or does not exist.The report that Fujino agriculture according to Obihiro and veterinary university teaches has the molecular species that surpasses 20 kinds of sphingolipids at least.The present invention includes these ceramides.

Content about LF among the present invention (hydrolyzate that comprises all LF, has LF derivant and the LF and the LF derivant of the physiologically active suitable) and ceramide with LF, the scope of estimating ceramide content preferably from 0.0001 to 0.1wt%, more preferably from 0.0005 to 0.01wt%, especially preferably from 0.0001 to 0.05wt%, all wt% are based on the gross weight of compositions, and the content range of estimation LF is preferably from 0.005 to 10wt%, especially preferably from 0.01 to 5wt%, most preferably from 0.01 to 3wt%, all wt% are based on the gross weight of compositions.Yet, believe that those skilled in the art can easily determine its optimum content in each objective composition, for example, by following test.Incidentally the content of the ceramide in Ruzhong is known, it is believed that it can be used as the standard of the ceramide amount that will add at least to a certain extent.

The present invention illustrates, and per os is taken in LF and suppressed antigenic specificity IgE production of antibodies.Therefore, those of ordinary skills can select to be suitable for the LF of preferred form of the present invention based on the present invention from above-mentioned various LF and effective dose thereof, for example by producing as an index with IgE antibody; The relation between clinical data, clinical symptoms etc. and the patients serum IgE of looking is determined dosage form and the effective dose of LF, be used for food, supplement, invalid's food, babies ' formula milk powder, health food, health requirements food, functional food, specific health food, medicine or and so on.Correspondingly, LF type and the effective dose of determining as mentioned above thereof comprises in the present invention.The technology that LF is added or be processed into food and so on is that know and commonly used., can use during at the LF that uses effective dose by it being configured to various dosage form well known by persons skilled in the art as medicine.

Embodiment

After this present invention will be described based on test and embodiment.But should remember that the present invention is not limited to these tests and embodiment.

In following test, with male immature BALB/c mouse (Japan SLC, the Inc. in 3 ages in week, Shizuoka Japan) is used for testing 1-4 and 6, with female BALB/c mouse (the Japan SLC in 8 ages in week, Inc., Shizuoka Japan) is used for testing 5.Carry out the significance test between each matched group and the corresponding test group thereof with student t check (Student ' s t test).

Test 1 N of LF (bLF) and suppress the activity that IgE antibody produces

At duration of test, allow LF use mice (n=7) in the group and arbitrarily take in a kind of casein containing protein as the feedstuff (based on AIN76) of protein source with contain the water of 1% N of LF (bLF, the product of DMV JapanK.K.).Allow mice (n=8) in the matched group arbitrarily take in the water of above-mentioned feedstuff and no bLF.

After on-test the 5th day and the 19th day carried out the intraperitoneal immunity with 10 μ g ovalbumins (OVA, the product of SEIKAGAKU CORPORATION) and 4mg aluminium hydroxide to every mice.At the 26th day, get blood from eye socket, measure total IgE antibody horizontal and OVA-specific IgE antibody level in the serum with ELISA.Total IgE antibody horizontal (Fig. 1) and OVA-specific IgE antibody level (Fig. 2) all are lower than matched group (* p＜0.05) in the bLF group mice serum.

Test 2 bLF and suppress the activity that IL-4 produces

In test 1, the excision spleen is with the preparation splenocyte suspension.Behind the haemolysis, in the presence of OVA, splenocyte was cultivated 72 hours in various concentration (5,10,50 and 100 μ g/mL).After the cultivation, measure IL-4 level in each culture supernatant with ELISA.Use in the group (n=7) at bLF, the IL-4 level under each OVA concentration all is lower than corresponding IL-4 level in the matched group (n=8).

Test 3 suppresses the activity of IgE antibody and cytokine generation through heat treated bLF

Heated 1 hour at 70 ℃ by the feedstuff [bLF (+)] that in the feedstuff of test 1, adds the 2.2%bLF acquisition a kind of.In contrast, will not add the feedstuff [bLF (-)] of bLF equally 70 ℃ of heating 1 hour.

At duration of test, allow mice arbitrarily take in bLF (+) (test group) or bLF (-) (matched group).After on-test the 5th day, the 14th day, the 23rd day and the 33rd day carried out the intraperitoneal immunity to every mice with ovalbumin (OVA, the product of SEIKAGAKU CORPORATION) (10 μ g) and aluminium hydroxide (4mg).Get blood from afterbody the 8th day (the 30th day) (7 age in week) after the 3rd peritoneum immunity, with the antibody in the ELISA mensuration serum.In addition, after the 3rd peritoneum immunity the 11st day (the 33rd day) and after the immunity of the 4th peritoneum the 8th day (the 40th day), depend on that each mice collects splenocyte respectively.The OVA of splenocyte and variable concentrations (0,50,100,200 and 400 μ g/mL) is being contained 10%FCS, 100U/mL benzylpenicillin, 100 μ g/mL streptomycins and 5 * 10 together ^-5Cultivated 72 hours in the RPMI1640 culture medium of M 2 mercapto ethanol.After the cultivation, measure IFN-in each culture supernatant with ELISA _γLevel and IL-4 level.

When carrying out 3 times when immune, the total IgE antibody horizontal in the test group serum is compared remarkable reduction (Fig. 4 and Fig. 5) with OVA-specific IgE antibody level with the matched group accordingly result.For splenocyte IFN-under each OVA concentration (0,50,100 and 200 μ g/mL) _γGeneration, between test group and matched group, do not observe substantial differences (Fig. 6).On the other hand, the generation of splenocyte IL-4 under each OVA concentration totally is lower than matched group in the test group, under the stimulation of 200 μ g/mL OVA, in the test group generation of IL-4 than matched group reduce more remarkable (Fig. 7).

When carrying out 4 times when immune, splenocyte is at each OVA concentration (0,50,100,200 and 400 μ g/mL) IFN-down _γGeneration, as the embodiment of 3 immunity the group between do not produce substantial differences (Fig. 8).On the other hand, in the test group under each OVA concentration the generation of IL-4 totally than matched group reduce more remarkable (Fig. 9).

By above result as seen, bLF inhibition antigenic specificity IgE antibody activity that produces and the activity that suppresses the IL-4 generation do not lost with heating.

Test 4 suppresses the activity of IgE antibody and cytokine generation through the bLF of hydrolysis

(1) preparation is through the bLF of hydrolysis

With trypsin " 207-09891 ", Wako Pure Chemical Industries, the product of Ltd., be used for the biochemistry purposes, from pig spleen preparation, 4,500USP trypsin unit/mg) be dissolved among the PBS of sterilization as trypsin stock solution (* 500,50mg/mL).With bLF (" 127-04122 ", Wako Pure Chemical Industries, the product of Ltd., lot.KSG7724) (10g) is dissolved in 25mM CaCl ₂Form 1% solution among the-0.1M Tris-HCl (pH8.2).After 1L bLF solution is heated to 37 ℃, add trypsin stock solution (2mL), then 37 ℃ of reactions 4 hours.After the reaction, heat 30 minutes so that enzyme deactivation at 80 ℃.After centrifugal 20 minutes of 1,700 * g, collect supernatant also with 0.45 μ m filter filtration sterilization, with " Micro Acilyezer S1 " (a kind of electrodialysis machine, by AsahiChemical Industry Co., Ltd. produces) handle sample, from sample, remove and desalt.Sample once more after the filtration sterilization, is stored in it-20 ℃, up to being used for test with 0.45 μ m filter.The yield of bLF hydrolyzate is 100% basically.The concentration of bLF hydrolyzate is 1%.

Allow mice arbitrarily take in the feedstuff and the solution that contains the 1%bLF hydrolyzate of test 1.Allow matched group arbitrarily take in same solid feed and the water of no bLF.After on-test the 5th day and the 19th day carried out the intraperitoneal immunity with ovalbumin (OVA, the product of SEIKAGAKU CORPORATION) (10 μ g) and aluminium hydroxide (4mg) to every mice.The 8th day (the 26th day) gets blood after the 2nd immunity, with the antibody in the ELISA mensuration serum.Simultaneously, depend on that also each mice collects splenocyte respectively.With splenocyte and OVA (0-400 μ g/mL) co-cultivation 72 hours, measure IFN-in each culture supernatant with ELISA _γWith the IL-4 level.

Total IgE antibody horizontal of observing in the test group serum is compared become lower (Figure 10 and 11) with OVA-specific IgE level with the respective horizontal in the matched group.

Notice that splenocyte produces IFN-under different OVA concentration _γGeneral inclination in test group, be higher than matched group (Figure 12).On the other hand, being created in of IL-4 do not demonstrate substantial differences (Figure 13) between test group and the matched group.

Test 5 LF to producing the effect of IL-12

Although IL-12 is a kind of cytokine of just finding relatively recently, known its acts on T cell and NK cell to induce IFN- _γGeneration (The Journal of Experimental Medicine (J.Exp.Med.), 173,869-879,1991; The Journal of Experimental Medicine (J.Exp.Med.), 177,1199-1204,1993).For the generation of inducing IL-12, the stimulation of cellular component such as LPS is effectively, and the cell that produces IL-12 comprises various kinds of cell, as macrophage, B cell and neutrophil.

The inventor cultivates peritoneal macrophages in the culture medium that adds bLF (Wako Pure Chemical Industries, the product of Ltd.), and measures IL-12 level in the culture supernatant with ELISA.

For the mice of random absorption feedstuff " MF " (Oriental Yeast Co., the product of Ltd), (2.5mL) respectively carry out peritoneal injection with thioglycollate medium (product of DIFCO Laboratories), to activate peritoneal macrophages.After 4 days, the Dulbeccos phosphate buffer (" Dulbeccos ' PBS ", NISSUI PHARMACEUTICALCO., the product of Ltd) that will contain 1%FCS is injected in the abdominal cavity with collecting peritoneum cell.After twice of cell washing, it is resuspended in " Dulbeccos ' PBS " in, add 96 orifice plates (2 * 10 ⁵Cell/0.2mL hole) afterwards, with plate incubation 2 hours in the CO2 incubator.Remove after the non-adherent cell, with containing 10%FCS, 100U/mL benzylpenicillin, 100 μ g/mL streptomycins, 5 * 10 ^-5RPMI 1640 culture medium (200 μ L) of M 2 mercapto ethanol and 100 μ g/mL bLF (Wako Pure Chemical Industries, the product of Ltd.) are filled with each hole.In contrast, use the above-mentioned culture medium of no bLF.With each plate incubation 18 hours in the CO2 incubator.After the cultivation, measure IL-12 level in each supernatant with ELISA.The result as shown in figure 14.Add the generation that bLF has significantly increased macrophage IL-12 in the culture medium.

From above result as seen, lactoferrin induces macrophage to produce IL-12.

Test 6 ceramides to IFN- _γThe effect of generation

Test use BALB/c mouse (3 ages in week, male) (Japan SLC, Inc.).Mice is divided into the lactoside ceramide is used group (test group) and the lactoside ceramide is not used group (matched group) (every group of 3 mices), so that the average of two groups of mice body weight and deviation equate basically.

As ceramide is that (product of Ltd. derives from milk, lotELK6191) to the lactoside ceramide for " biological chemical reagent 126-04491 ", Wako Pure Chemical Industries.The concentration of lactoside ceramide with 200 μ g/mL is dispersed in the 0.9%NaCl solution that contains 0.5%Tween20, and with dispersions obtained being stored in-20 ℃ up to use.In use, dispersion is thawed, to the aseptic PBS that goes out that wherein adds equivalent.After fully mixing, give in the mouse peritoneum and used lactoside ceramide solution (every mice 200 μ L) totally 14 days every day, except Sunday and Saturday (every mice 20 μ g).For matched group, only use solvent (contain the 0.9%NaCl solution of 0.5%Tween20 and add the sterilized PBS of equivalent).

The splenocyte that will obtain from every mice in the 14th day after administration is seeded to (" No.3072 " produced by Becton Dickinson and Company) the 96 hole microtest plates, and inoculum concentration is every hole 4 * 10 ⁵Cell.Cultivating the culture medium that adopts is RPMI 1640 culture medium (" No.11875-093 ", the product of Gibco BRL), wherein contain 10% hyclone (product of JapanBiotest Laboratory, lot 10086-1), 100U/mL penicillin, 100 μ g/mL streptomycins and 5 * 10 ^-5The M 2 mercapto ethanol.Cultivation is carried out 4 and is taken turns, and splenocyte was cultivated 20 hours down in the existence of bLF (product of MeggleGmbH, 0 or 100 μ g/mL).After the cultivation, measure the IFN-in each culture supernatant with ELISA test kit (product of ENDOGEN Inc.) _γWith the two-way classification variance analysis (2 * 2, significance level: analytical data 5%), get and use lactoside ceramide and bLF and stimulate as factor.

The result as shown in figure 15.When not using ceramide, whether bLF exists is not all observed IFN- _γGeneration.When using ceramide in advance, bLF do not exist and in the presence of all observe IFN- _γGeneration significantly increase.Owing to observe interaction (p=0.0016) between ceramide and these two factors of bLF, finding to use the lactoside ceramide increases the IFN-that cattle LF stimulates _γProduce.

Embodiment 1

Cattle LF is added " EPITOLESS " (MEIJI MILK PRODUCTSCO., product of LTD) with 0.01% concentration, and this is a kind of breast at newborn autopath, and it does not use lactalbumin as raw material, and has following prescription.

Composition		Per 100 gram products
			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
			Nicotinic acid	mg	6
Folic acid	mg	0.2
			Beta-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
Ferrum	mg	6.5
			Copper	μg	320
Zinc	mg	2.8

Embodiment 2

Cattle LF is added through breast " MEIJI NOBIYAKA " the MEIJIMILK PRODUCTS of hydrolysis CO., the product of LTD with 0.01% concentration), it contains lactalbumin as raw material.

Composition		Per 100 gram products
			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
			Nicotinic acid	mg	6
Folic acid	mg	0.2
			Beta-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
Ferrum	mg	6.5
			Copper	μg	320
Zinc	mg	2.8

Industrial applicability

Find that by the present invention per os is taken in the generation that LF suppresses IgE antibody in the body.

Claims (4)

1. a mammalian antigen specific IgE antibody that comprises the people produces inhibitor, and it comprises lactoferrin or its hydrolyzate and ceramide as active component.

2. lactoferrin or its hydrolyzate and ceramide are used to prepare the purposes that the mammalian antigen-specific IgE antibody that comprises the people produces inhibitor.

3. the purposes of the compositions of immunity is controlled in the generation that is used to prepare the mammalian antigen-specific IgE antibody by suppressing to comprise the people of lactoferrin or its hydrolyzate and ceramide.

4. lactoferrin or its hydrolyzate and ceramide are used to prepare and are used to comprise that people's mammiferous IFN-γ produces the purposes of enhancer compositions.

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