JP5935200B2 - Immunostimulants from helminth parasites - Google Patents

Description

  The present invention relates to a Th2 immunostimulant comprising a desurfactant extracted fraction obtained by removing a surfactant from a surfactant fraction extracted from a parasite using a surfactant and a method for producing the immunostimulant. Etc.

  In an immune reaction, functioning helper T cells (Th) are mainly classified into type I (Th1) cells and type II (Th2) cells (for example, Non-Patent Document 1, Non-Patent Document 16, Non-Patent Document 17). Among them, Th1 cells are known to be involved in cellular inflammatory reactions. Examples of such cellular inflammatory reactions include IL-2 and IFN-γ, which are cytokines secreted by Th1 cells (Th1 cytokines). Induced cytotoxic reactions, delayed hypersensitivity reactions and the like are known. On the other hand, Th2 cells are known to be involved in humoral immune responses. Such humoral immune responses are induced by IL-4 and IL-5, which are cytokines secreted by Th2 cells (Th2 cytokines), for example. Allergic reactions associated with increased IgE levels, defense reactions from parasites, and the like are known (see, for example, Non-Patent Document 17). Further, it is known that the type of immune reaction is rapidly changed to either Th1 type or Th2 type by inhibiting the action of the other cytokine by Th1 cytokine and Th2 cytokine (for example, non-patent literature). 1 and non-patent document 16). Such an immune reaction system induces an appropriate immune response in vivo according to the type of foreign antigen (see, for example, Non-Patent Document 1 and Non-Patent Document 16), but the balance control of the immune reaction is disturbed, and the Th1 type It is known that excessive tilting causes tissue damage diseases such as Crohn's disease and organ-specific autoimmune diseases such as rheumatoid arthritis and autoimmune hepatitis (for example, Non-Patent Document 1 and Non-Patent Document 7). Non-patent document 16). On the other hand, it is known that excessively tilting to Th2 type causes immune diseases such as allergies, atopy, and systemic autoimmune diseases (see, for example, Non-Patent Document 1 and Non-Patent Document 16). In order to treat these diseases, research aimed at restoring the balance of disturbed immune responses to normal is being conducted.

  Since LPS (Lipopolysaccharide) and CpG (cytosine-phosphate-guanine) can induce the activation of Th1 immune response even after single administration (see, for example, Non-Patent Document 14), the balance that is excessively inclined to Th2 type is Th1. It is attracting attention as a tool to return to the mold side. As one of the processes in the activation of immune response (immunostimulation), after antigen is taken up by antigen-presenting cells, the antigen-treated peptide fragment binds to MHC (major histocompatibility antigen complex) molecule by processing Then, it is known that Th cells recognize antigens expressed on the cell surface of antigen-presenting cells, and sensitization occurs (for example, Non-Patent Document 1, Non-Patent Document 6), but Th1 immunization with LPS or CpG The activation of the reaction (Th1 immunostimulation) is different from the above-mentioned process, because LPS and CpG directly stimulate antigen-presenting cells as ligands of TLR (Tall-like receptor) and cause an immune reaction in the host. It is known that CpG is highly useful as a substance having Th1 immunostimulatory activity (Th1 activator). In addition, since the substance having Th1 immunostimulatory activity can be Th1 immunostimulatory by itself, it is suitable for basic research to elucidate the mechanism of Th1 immune reaction, for example, research to elucidate intracellular stimulation transmission pathways in antigen-presenting cells. Is also known to be applied (see Non-Patent Document 14, for example).

  On the other hand, a search for a substance having Th2 immunostimulatory activity has also been conducted, and among them, for example, Pam-3-cys which is an insoluble substance (lipopeptide) present on the surface of mycoplasma cells (see, for example, Non-patent Document 3). ) And FSL-1 (see, for example, Non-Patent Document 4) have been reported to activate Th2 immunostimulation by directly acting on antigen-presenting cells via TLR2 (Tall-like receptor 2). On the other hand, these substances have the effect of increasing the production of Th1 cytokines (IL-6 and the like) in addition to Th2 cytokines (see, for example, Non-Patent Document 14), and in vitro experimental systems are used for Th2 immunostimulation. Also requires a high dose (see, for example, Non-Patent Document 4), and the effect of Th2 immunostimulation is not sufficient (see, for example, Non-Patent Document 14) And it is also known. Furthermore, research has been conducted to search for substances having Th2 immunostimulatory activity from gastrointestinal parasites that cause strong Th2 immunostimulation. As a result, some substances that activate Th2 immunostimulatory have been identified. These substances require an adjuvant as a mere antigen, require sensitization / challenge by frequent administration (see, for example, Non-Patent Document 6), or are effective only in vitro (for example, non-patent) Reference 8) is known.

Mosmann TR, Sad S. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more.Immunol Today. 17 (3): 138-46. Bordier C. 1981.Phase separation of integral membrane proteins in TritonX-114 solution.J Biol Chem. 256 (4): 1604-7. Hasebe A, Mu HH, Washburn LR, Chan FV, Pennock ND, Taylor ML, Cole BC. 2007. Inflammatory lipoproteins purified from a toxigenic and arthritogenic strain of Mycoplasma arthritidis are dependent on Toll-like receptor 2 and CD14. Infect Immun. 75 (4): 1820-6. Kiura K, Kataoka H, Yasuda M, Inoue N, Shibata K. 2006. The diacylated lipopeptide FSL-1 induces TLR2-mediated Th2 responses. FEMS Immunol Med Microbiol. 48 (1): 44-55. N. Ishikawa, Y. Horii, Y. Nawa. 1994. Inhibitory effects of concurrently present ‘normal’ Nippostrongylus brasiliensis worms on expulsion of ‘damaged’ worms and associated goblet cell changes in rats. Parasite Immunology. 16: 329-332. Wilson MS, Elnekave E, Mentink-Kane MM, Hodges MG, Pesce JT, Ramalingam TR, Thompson RW, Kamanaka M, Flavell RA, Keane-Myers A, Cheever AW, Wynn TA. 2007. IL-13Ralpha2 and IL-10 coordinately suppress airway inflammation, airway-hyperreactivity, and fibrosis in mice.J Clin Invest. 117 (10): 2941-51. R W Summers, D E Elliott, J F Urban Jr, R Thompson, J V Weinstock. 2005. Trichuris suis therapy in Crohn ’s disease. Gut. 54: 87-90. Li Q, Kumar A, Gui JF, Yu FS. 2008. Staphylococcus aureuslipoproteins trigger human corneal epithelial innate response through toll-like receptor-2. Microb Pathog. 44 (5): 426-34. Metcalfe DD.1984. Mast cell mediators with emphasis on intestinal mast cells. Ann Allergy. 53: 563-75. McKenzie GJ, Bancroft A, Grencis RK, McKenzie AN. 1998. A distinct role for interleukin-13 in Th2-cell-mediated immune responses. Curr Biol. 8 (6): 339-42. Perdue MH, Ramage JK, Burget D, Marshall J, Masson S. 1989. Intestinal mucosal injury is associated with mast cell activation and leukotriene generation during Nippostrongylus-induced inflammation in the rat.Dig Dis Sci. 34 (5): 724-31 . Holland MJ, Harcus YM, Riches PL, Maizels RM. 2000. Proteins secreted by the parasitic nematode Nippostrongylus brasiliensis act as adjuvants for Th2 responses. Eur J Immunol. 30 (7): 1977-87. Dullemen HMV. 1994. Treatment of Crohn ’s disease with anti-tumor necrosis factor chimeric antibody (cA2). Gastroentrerol. 109: 129-135. Dillon S, Agrawal A, Van Dyke T, Landreth G, McCauley L, Koh A, Maliszewski C, Akira S, Pulendran B. 2004. A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells. J Immunol. 172 (8): 4733-43. Kamiya M, Oku Y, Itayama H, Ohbayashi M. 1985. Prolonged expulsion of adult Trichinella spiralis and eosinophil infiltration in mast cell-deficient W / Wv mice. J Helminthol. 59 (3): 233-9. Carter LL, Dutton RW. 1996. Type 1 and type 2: a fundamental dichotomy for all T-cell subsets. Curr Opin Immunol. 8 (3): 336-42. Urban JF Jr, Madden KB, Svetic A, Cheever A, Trotta PP, Gause WC, Katona IM, Finkelman FD. 1992. The importance of Th2 cytokines in protective immunity to nematodes. Immunol Rev. 127: 205-20.

  At present, a substance having a sufficient effect of Th2 immunostimulatory activity for use in vivo has not yet been found. Therefore, the search for a substance having Th2 immunostimulatory activity is not only a therapeutic agent that restores the balance of immune response leaning to Th1 side to Th2 side, but also elucidates the mechanism in the early stage of Th2 immunostimulation, preventing allergy, It is also considered important for obtaining knowledge for therapeutic drug development. An object of the present invention is to provide a Th2 immunostimulator that causes strong Th2 immunostimulation in vivo.

  Researchers in this field have focused on the Th2 immunostimulatory action of gastrointestinal helminth parasites, and have studied exclusively the Th2 immunostimulatory action of water-soluble extract fractions of gastrointestinal helminth parasites (for example, non-patented) Reference 8). However, the present inventor considered that there is a limit to practical use in the Th2 immunostimulatory action of the water-soluble extract fraction of gastrointestinal helminth parasites. Recently, Nb was collected from a rat infected with Nippostrongylus brasiliensis (hereinafter sometimes referred to as “Nb”), a kind of gastrointestinal helminthic parasite, and Nb was collected in another rat. In experiments to infect Nb, it was reported that Nb physically damaged at the time of exclusion caused Th2 activation reaction earlier than Nb collected at the early stage of infection, and Nb was eliminated (for example, non-existing). Patent Document 5). From these results, the present inventor considers that the component leaked from Nb may have a high Th2 immunostimulatory effect, and in order to search for the component, Nb is considered to be disadvantageous in purification and subsequent formulation. It was decided to examine the poorly water-soluble fraction. The Nb-derived surfactant fraction (poorly water-soluble fraction) extracted using TritonX-114, which is one of the surfactants, has a high Th2 immunostimulatory effect that has not been obtained in the prior art. As a result, the present invention has been completed.

That is, the present invention provides (1) Nippo stolons di Las brasiliensis emissions cis (Nippostrongylus brasiliensis) consist solely de-detergent extraction fraction to remove the surfactant from the surfactant fraction extracted using a surfactant from Th2 adjuvant, wherein (excluding food) or, (2) removing detergent extraction fraction, Th2 or immunostimulant as described in (1) above, characterized in that it comprises a peptide, (3) The Th2 immunostimulator according to (1) or ( 2 ) above, wherein the surfactant is TritonX-114 (registered trademark), and (4) the presence or absence of Th2 immunostimulatory activity, The Th2 immunostimulator according to any one of (1) to (3) above, wherein the determination is based on an increase in the number of acid spheres or an increase in the expression level of IL-4, and (5) a form of oral administration (1) ) To (4), the Th2 immunostimulator.

In addition, the present invention provides (6) a Th2 immunostimulator according to any one of (1) to (5) above, a pharmaceutically acceptable normal carrier, binder, stabilizer, excipient, and diluent. , A Th2 immunostimulatory composition to which a pH buffer, a disintegrant, a solubilizer, a solubilizer, or an isotonic agent is added.

The present invention further includes (7) (a) a step of mixing Nippostrongylus brasiliensis or a treated product thereof with a surfactant-containing aqueous solution; (b) a water layer and a surfactant layer. (C) a step of collecting a surfactant fraction; (d) a step of removing the surfactant from the surfactant fraction to obtain only a desurfactant extracted fraction; A method for producing a Th2 immunostimulator according to (1) above, or (8) a treated product of Nippostrongiras bragiliensis, characterized in that a physically destroyed treated product of Nippostrongillus brasiliensis Or (9) the production method according to (7) or (8), wherein the surfactant is TritonX-114 (registered trademark) And (10) in step (d) Removal process surfactants, a method of manufacturing according to any one of the above, which comprises a precipitation treatment of the protein (7) to (9).

  According to conventional techniques (for example, see Non-Patent Document 6 and Non-Patent Document 8), substances that cause Th2 immunostimulation are those that require an adjuvant as a simple antigen, those that require sensitization / challenge by frequent administration, However, according to the Th2 immunostimulant of the present invention, the Th2 immunostimulator by a single oral administration, particularly immunity with few side effects that does not require the use of an adjuvant. It can be expected to lead to the development of activators and vaccines, and is also expected to be a useful tool for basic research to elucidate the mechanism at the early stage of Th2 immunostimulation.

It is a figure which shows the body weight of each group on the 7th and 14th day after administration of Th2 immunostimulator (TX) of this invention, a parasite all component (AW), or PBS. It is a figure which shows the small intestine weight (g / BWgx100) of each group in the 7th and 14th day after administration of TX, AW, or PBS. It is a figure which shows the spleen weight (g / BWgx100) of each group in the 7th and 14th day after administration of TX, AW, or PBS. It is a figure which shows the mesenteric lymph node weight (g / BWg * 100) of each group in the 7th and 14th day after administration of TX, AW, or PBS with a graph. It is a figure which shows the eosinophil count in the peripheral blood of each group on the 7th and 14th day after administration of TX, AW or PBS. It is a figure which shows the IgE density | concentration in the serum of each group on the 7th and 14th day after administration of TX or PBS. It is a figure which shows the IL-4 mRNA expression level (PBS administration group shall be 1.00) in the spleen and mesenteric lymph node of each group on the 7th day after the administration of TX or PBS. The vertical axis represents the exponent value of 2. It is a figure which shows the result of the optical microscope (160 time) observation of the small intestine (HE dyeing | staining section | slice) 7 days after administration in a PBS administration group. It is a figure which shows the result of the optical microscope (160 time) observation of the small intestine (Alcian blue dyeing | staining section | slice) 7 days after administration in a PBS administration group. It is a figure which shows the result of the optical microscope (160 time) observation of the small intestine (HE dyeing | staining section | slice) 7 days after administration in an AW group. It is a figure which shows the result of the optical microscope (160 time) observation of the small intestine (Alcian blue dyeing | staining section | slice) 7 days after administration in an AW group. It is a figure which shows the result of the optical microscope (160 time) observation of the small intestine (HE dyeing | staining section | slice) 7 days after administration in a TX group. It is a figure which shows the result of the optical microscope (160 time) observation of the small intestine (alcian blue dyeing | staining section | slice) 7 days after administration in TX group. It is a figure which shows the eosinophil count in the peripheral blood of each group of the 3rd and 5th day after administration in TX group, SDS processing TX group, or ProK processing TX group.

  As the Th2 immunostimulant of the present invention, as long as it contains, as an active ingredient, a desurfactant extracted fraction obtained by removing a surfactant from a surfactant fraction extracted from a parasite using a surfactant. The method for producing the Th2 immunostimulator of the present invention is not limited, and the step (a) of mixing a parasite or a processed product thereof with a surfactant-containing aqueous solution; a layer of an aqueous layer and a surfactant layer Step (b) for performing a separation treatment; Step (c) for collecting a surfactant fraction; Step (d) for obtaining a desurfactant-extracted fraction by performing a treatment for removing the surfactant from the surfactant fraction; The “Th2 immunostimulant” herein refers to a substance having an action of activating a Th2 immune reaction.

  Examples of the parasites include protozoan parasites composed of single cells such as sporeworms, rhizopods and flagellates, and helminth parasites composed of multiple cells such as nematodes, flukes and tapeworms. However, helminth parasites are preferred because Th2 immunostimulation occurs upon infection with helminth parasites. Of the helminthic parasites, examples of helminthic parasites that infect mice include Trichuris muris, Trichinella spiralis, Nippostrongylus brasiliensis, Heligmonsomoides polygyrus, Hymenolepsis nana, and the like. Examples of helminth parasites that infect worms include Angiostrongylus, and examples of helminth parasites that infect pigs include Trichuris suis, Ascaris suum, and worms that infect dogs and cats. Examples of sexual parasites include Trichuris vulpis, Toxocara, Gnathostoma, and Ancylostoma. Examples of helminth parasites that infect marine mammals include Anisakis and Pseudoterranova, which infect birds. Examples of helminthic parasites include S. douthitti, Trichobilharzia ocellata, T. stagnicolae, T. physellae, Gigantobilharzia hur onensis etc. can be mentioned, and among these, Nippostrongiras bragiliensis can be preferably exemplified.

  Examples of the surfactant include nonionic surfactants, amphoteric surfactants, and ionic surfactants. Among these, nonionic surfactants include, for example, TritonX-100 and TritonX. -114, NP-40, Tween-20, Tween-80 and the like. Examples of amphoteric surfactants include 7BzO, SB3-10, SB3-14, CHAPS, and amidosulfobetaine-14 (ASB14). Examples of the ionic surfactant include cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). Among these, the cloud point is about 20 ° C. TritonX-114 is preferable because it can be separated into an aqueous solution fraction and a surfactant fraction under typical conditions.

  The above-mentioned desurfactant-extracted fraction may contain, for example, polynucleotides, peptides, lipids, carbohydrates and the like, which are constituents of parasites. Among these, the Th2 immunostimulatory activity can be obtained by protease treatment. Since it is lost, it is highly possible that peptides are included. Such a desurfactant-extracted fraction can be prepared as follows.

  First, a parasite or a processed product thereof and a surfactant-containing aqueous solution are mixed. As a method for obtaining the processed product of the parasite, a treatment method in which the parasite is destroyed so that the surfactant-containing aqueous solution can easily penetrate before mixing the parasite and the surfactant-containing aqueous solution is preferable. For example, biochemical destruction treatment, physical destruction treatment, and the like can be mentioned, and physical destruction treatment that can effectively and efficiently destroy the parasite is preferable. Examples of the treatment method in the physical destruction treatment include a freeze-thaw method, an ultrasonic crushing method, a homogenizer crushing method, a glass bead crushing method, etc. Among them, the ultrasonic crushing treatment method is preferably exemplified. can do.

  The surfactant-containing aqueous solution may be an aqueous solution containing the surfactant, and the concentration of the surfactant may vary depending on the type of surfactant used (for example, Molecular Cloning). For example, when TritonX-114 is used, it is preferably 0.2% to 10%, more preferably 1% to 3%. Moreover, you may add a buffer, a proteolytic enzyme inhibitor, a preservative, etc. as needed.

  Next, a layer separation process is performed between the aqueous layer and the surfactant layer. As such a layer separation treatment, a treatment using a phenomenon in which micelle formation cannot be achieved when a surfactant-containing aqueous solution is raised from a temperature lower than its cloud point to a higher temperature, and separation into an aqueous layer and a surfactant layer is preferable. When TritonX-114 is used as the surfactant, the cloud point is about 20 ° C., so the temperature lower than the cloud point is 0 ° C. to 20 ° C., preferably 2 ° C. to 10 ° C., higher than the cloud point. Examples of the temperature include 20 ° C to 42 ° C, preferably 30 ° C to 40 ° C.

  Subsequently, the surfactant fraction is collected. Such a collecting method may be any method that can collect a surfactant layer as a surfactant fraction from a surfactant-containing aqueous solution separated into an aqueous layer and a surfactant layer. Since the specific gravity is heavier than that of the aqueous layer, the surfactant layer is separated below the aqueous layer. Therefore, a method of removing the aqueous layer by collecting the aqueous layer by decantation or pipette can be used.

  Next, a surfactant is removed from the surfactant fraction to obtain a desurfactant extracted fraction. Examples of the method for removing the surfactant from the surfactant fraction include a method under non-denaturing conditions using a column, an ultrafiltration membrane, etc., methanol precipitation, ethanol precipitation, acetone precipitation, TCA (trichloroacetic acid) precipitation, etc. And the like. However, since the Th2 immunostimulatory activity does not disappear even under denaturing conditions, considering the ease of operation and cost effectiveness, the method is carried out under denaturing conditions. Is preferred. As described above, when the surfactant is removed from the surfactant fraction, a desurfactant extracted fraction that is an active ingredient of the Th2 immunostimulator of the present invention can be obtained.

  As a method for confirming that the above-mentioned desurfactant-extracted fraction has Th2 immunostimulatory activity, for example, a substance induced (increased) by Th2 immunostimulation, a substance suppressed (decrease) by Th2 immunostimulatory, etc. Can be mentioned, and among these, it is preferable to carry out using as an index a substance that increases by Th2 immunostimulation. Examples of substances that increase by Th2 immunostimulation include IgE, eosinophils, Th2 cytokines (IL-4, IL-5, IL-9, IL-10, IL-13, etc.), and the like. Among these, IgE, eosinophils, and IL-4 can be preferably exemplified.

  The composition having the Th2 immunostimulatory activity of the present invention is not particularly limited as long as it contains the Th2 immunostimulator of the present invention, and the case where the Th2 immunostimulator of the present invention is used as a therapeutic agent for pharmaceuticals, etc. Normal carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents, etc., pharmaceutically acceptable for Th2 immunostimulators Compositions to which various ingredients for preparations are added and compositions to which ingredients such as preservatives, antioxidants, colorants, sweeteners are added, such as when the Th2 immunostimulant of the present invention is used as a supplement, etc. Things can be exemplified.

  Examples of the dosage form of the Th2 immunostimulator and the composition having Th2 immunostimulatory activity of the present invention include oral administration in a dosage form such as powder, granule, tablet, capsule, syrup, suspension, solution, Examples include parenteral administration in which dosage forms such as emulsions and suspensions are injected or administered intranasally in the form of sprays, but a single oral administration has sufficient Th2 immunostimulatory effect, so oral administration is preferable. Further, the Th2 immunostimulatory agent and the composition having Th2 immunostimulatory activity of the present invention are pharmaceuticals useful for tissue disorder diseases such as Crohn's disease, organ-specific autoimmune diseases such as rheumatoid arthritis and autoimmune hepatitis. Can be used as a supplement, functional food. Such functional foods include yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, tea, sencha, oolong tea, sports drinks and other beverages, pudding, cookies, bread, cakes, jellies, rice crackers, etc. Japanese sweets such as confectionery, sheep candy, frozen confectionery, bread and confectionery such as chewing gum, noodles such as udon and soba, fish paste products such as kamaboko, ham and fish sausage, miso, soy sauce, dressing, mayonnaise, sweeteners, etc. Examples include seasonings, dairy products such as cheese and butter, and various side dishes such as tofu, konjac, other boiled rice cakes, dumplings, croquettes, and salads.

  Hereinafter, the present invention will be specifically described by way of examples. However, the technical scope of the present invention is not limited by the examples. In the examples, all data are shown as mean ± standard error (SE), and the test of significant difference using statistical analysis was performed by Mann-Whitney U-test. Differences between means were significant when P-value was less than 0.05. In this example, SD rats (Slc) or Wistar rats (Slc) were used as rats, and ICR mice (Slc) (female, 8 weeks old, SPF) were used as mice. In addition, as a method for raising rats in this example, a sample (Nihon Clare, CE-2, Osaka, Japan) that was heat sterilized at 120 ° C. for 20 minutes in a plastic cage in which wood chips (sterilized) were laid until dissection. As a breeding method of mice, the feed was heat-sterilized at 120 ° C. for 20 minutes in a metal cage (both heat-sterilized) with wood chips (Japan Claire, CE-2, (Osaka, Japan) and a method of freely taking tap water. The method for raising the rats and mice was carried out in accordance with the animal experiment guidelines of the Faculty of Agriculture, Yamaguchi University.

[Nb passage method and recovery method]
Nippostrongiras bragiliensis (Nb) (distributed by Tokyo Jikei University School of Medicine) was recovered after being subcultured and expanded using rats. After collecting Nb3500-4000 vessels / animal in the third stage larvae (L3) by the Baermann method (Step 1), Nb was inoculated subcutaneously into the groin area of the rat, and the eggs discharged from Nb that became adults 8 days later Intestinal stool and cecal stool were collected (step 2). Thereafter, the eggs discharged from Nb were grown to L3 by performing a charcoal culture method (step 3). By repeating step 1 to step 3 again, Nb was passaged and propagated. The recovery of Nb was performed by the procedure described below by the Baermann method. That is, the small intestine of a rat 8 days after Nb inoculation was collected, cut vertically, and then placed on a gauze clipped so as to cover the mouth of a 200 ml metric glass, 0.9% physiological saline (PBS And was allowed to stand at 37 ° C. for 1 hour so that Nb was filtered. After that, Nb sinking to the bottom was collected together with a small amount of physiological saline, transferred to a funnel on which filter paper was placed, passed through about 80 ml of physiological saline several times, filtered and washed, and Nb was recovered.

[Method for obtaining fraction of desurfactant by TritonX-114]
After adding 1 ml of TE (Tris-EDTA Buffer) to about 2000 adult Nb recovered in Example 1 above, ultrasonic crushing treatment was performed using BRANSON SONIFIER 450. 0.25 ml of 10% TritonX-114 (Sigma-Aldrich) solution was added to the ultrasonically crushed product obtained by the above treatment, and the mixture was allowed to stand at 4 ° C. for 2.5 hours, and then centrifuged (13,000 rpm). The supernatant is collected by 3 minutes, 4 ° C., and then subjected to layer separation by stationary treatment (37 ° C., 10 minutes) and centrifugation (13000 rpm, 5 minutes, 24 ° C.) (for example, Non-Patent Document 2). In addition, 1 ml TE and 2.5 times the amount of ethanol were added to the lower layer (surfactant layer) from which the upper layer (aqueous layer) was removed, and the mixture was then allowed to stand at −4 ° C. overnight and centrifuged. The precipitate (desurfactant fraction) obtained by the treatment (3000 rpm, 20 minutes, 4 ° C.) was suspended in 0.2 ml PBS.

[Oral administration of desurfactant fraction to mice]
After the protein amount of the desurfactant fraction obtained in Example 2 above was measured using the Bradford method, the desurfactant fraction diluted with olive oil so that the protein concentration was 150 μg / 500 μl was added to the mouse. The group to be orally administered to was designated as a desurfactant fraction administration (TX) group. As a control, a group in which PBS was orally administered to mice instead of the desurfactant fraction was defined as a PBS administration (PBS) group. Furthermore, the ultrasonic disruption product obtained by adding 1 ml of TE to about 2000 adult Nb and subjecting to ultrasonic disruption was diluted with olive oil to 150 μg / 500 μl. Was orally administered to mice as Nb administration (AW) group. That is, oral (intragastric) administration using a sonde was performed in a total of three groups, a TX group, a PBS group, and an AW group.

  After oral administration to mice, the body weight (g) of each group was measured on the 7th and 14th. As a result, no significant difference was observed in the body weight in each of the PBS group, AW group and TX group (Fig. 1).

  Known biological reactions observed during gastrointestinal parasitic helminth infection include increases in mast cells and goblet cells in the intestinal tract, increased mucus secretion from goblet cells, and swelling of mesenteric lymph nodes and Peyer's patches. For example, refer nonpatent literature 17). In the small intestine of Nb-infected individuals, edema occurs due to leukotriene and heparin produced by activated mucosal mast cells (see, for example, Non-Patent Document 9), increased permeability of the intestinal epithelium by histamine release, etc. (for example, Non-Patent Document 11). The increase in the number of mast cells is considered to be caused by the increase in production of Th2 cytokine (for example, see Non-Patent Document 11). Therefore, if the Th2 immune reaction is activated by oral administration of the parasite-derived desurfactant fraction, edema occurs in the mouse small intestine of the TX group, and the weight of the small intestine increases. is expected. Therefore, the weight of the small intestine was measured (FIG. 2). In addition, the weights of the spleen and mesenteric lymph nodes were also measured as a comparative control (FIGS. 3 and 4). After oral administration, the weight of the small intestine (g / BWg × 100) was significantly less in the TX group than in the PBS group or AW group on both the 7th and 14th days (FIG. 2). Further, regarding the spleen weight (g / BWg × 100) and the mesenteric lymph node weight (g / BWg × 100), no significant difference was observed in each of the PBS group, AW group and TX group (FIG. 3). And FIG. 4).

  Furthermore, histopathological observation was performed in order to examine whether or not tissue change due to inflammation or the like caused by Th2 immunostimulation was observed in the small intestine. A histopathological specimen was prepared by the following procedure. That is, in each group of PBS group, AW group, and TX group, mice were anesthetized with ether after 7 or 14 days after oral administration, and then euthanized by whole blood collection using a heparinized syringe, Thereafter, the jejunum was collected from the spleen, mesenteric lymph node, liver, and duodenum base, and then fixed with Carnoy's solution, and then a 4 μm paraffin section (pathological tissue specimen) was prepared. As a result of observing the histopathological specimen with a conventional hematoxylin-eosin (HE) staining and Alcian blue staining with an optical microscope, no edema of the lamina propria due to parasitic infection was observed ( 8 to 13). In addition, since Th2 cytokines have been reported to increase the number of obese (mast) cells and goblet cells by Th2 immunostimulation, the number of mast cells and goblet cells in the above pathological tissue specimen can be determined for each mouse. The average value was obtained by counting 10 or more units with villi-crypt as 1 unit, and the number of cells per villi of the small intestine mucosa (cell number / villi crypt unit) was calculated. Compared to 12, there was little difference in the TX group with 0.16, and similarly, goblet cells showed little difference with 4.2 in the TX group versus 4.1 in the control group.

  From histopathological observations, it was not known whether Th2 immunostimulation occurred or not. As another index for verifying the presence of Th2 immunostimulation, an increase in IgE amount and an increase in eosinophil count , And an increase in IL-4 mRNA level was used for analysis. In order to measure the amount of IgE, serum prepared from peripheral blood collected from each group by centrifugation (12000 rpm, 30 minutes) was stored frozen at −80 ° C. and then mouse IgE measurement kit (Morinaga Institute of Science) Was analyzed by the EIA sandwich method (FIG. 6). As a result, the IgE concentration (ng / ml) in the peripheral blood 7 days after oral administration was not significantly different between the PBS group and TX group, whereas it was 14 days after oral administration. The IgE concentration was 4.3 times higher in the TX group than in the PBS group, and was significantly increased (FIG. 6).

In addition, in order to measure the number of eosinophils, a part of blood collected by cardiac blood sampling is diluted 10 times with Hinkelmann's solution (0.5% yellow eosin, 0.5% phenol, 0.5% formalin) After gently stirring, the number of eosinophils was counted using an eosinophil counter (TATAI) (FIG. 5). As a result, the number of eosinophils in the peripheral blood 7 days after oral administration (eosinophil count / 0.1 mm ³ ) was 2.4 times higher in the AW group than in the PBS group, compared to the PBS group. The TX group increased by 6.8 times, and the TX group increased by 2.9 times compared with the AW group. On the other hand, on day 14 after oral administration, the number of eosinophils in the TX group (eosinophil count / 0.1 mm ³ ) decreased compared to 7 days, and no significant difference was observed with the PBS group (Fig. 5).

Furthermore, in order to detect the amount of IL-4 mRNA, RNA extraction, cDNA synthesis, and mRNA detection by quantitative PCR were performed according to the following procedures. That is, RNA was extracted according to the manual using RNeasy ^™ Kit (manufactured by QIAGEN) from part of the spleen of mice in the TX group and PBS group 7 days after oral administration and mesenteric lymph nodes. Subsequently, 1 μg of a random primer (Invitrogen) was added to 10 μl of the extracted RNA, and immediately left on ice at 70 ° C. for 10 minutes, and then 5 × RT buffer, dNTP, 0.1 M DTT, RNAsin (Promega) Was added to make 19 μl, and 1 μl of SuperScript II (manufactured by Invitrogen) was added for 2 minutes at 42 ° C. to give a total of 20 μl, which was reacted at 42 ° C. for 50 minutes and 70 ° C. for 15 minutes. Further, Step One ^™ Real-Time using a synthesized cDNA, a primer set for amplifying IL-4 gene product (IL-4 mRNA) (Applied Biosystems), and TaqMan ^™ Gene Expression Assays kit (Applied Biosystems). Real-time PCR was performed by PCR System (Applied Biosystems) to quantify the amount of IL-4 mRNA. PCR conditions were 50 ° C. for 2 minutes, 95 ° C. for 10 minutes for 1 cycle, 95 ° C. for 15 seconds, and 60 ° C. for 1 minute for 50 cycles. As a control, a real-time PCR method using a primer set (manufactured by Applied Biosystems) that amplifies 18s rRNA mRNA that is a housekeeping gene was performed, and the value of IL-4 mRNA was standardized by the calculated value of 18s rRNA mRNA. Further, the value of IL-4 mRNA in the TX group was calculated when the value of IL-4 mRNA in the PBS group was 1 (FIG. 7). As a result, in the spleen, there was no significant difference in the amount of IL-4 mRNA in the TX group compared to the PBS group. On the other hand, in the mesenteric lymph node, IL-4 mRNA expression in the TX group was increased 128-fold compared with the PBS group, and a significant difference was observed (FIG. 7).

  In order to examine whether the active ingredient of the desurfactant fraction is a peptide (including protein), SDS was used as a protein denaturing agent and ProK (manufactured by Sigma) as a proteolytic enzyme. That is, after the desurfactant fraction was treated in a 0.5% SDS / TE solution or a 0.001% ProK / (0.5% SDS / TE) solution at 37 ° C. for 12 hours. , 2.5 times the amount of ethanol, and then allowed to stand at −4 ° C. overnight, and then the precipitate obtained by centrifugation (3000 rpm, 20 minutes, 4 ° C.) was suspended in 0.2 ml PBS. . Of these precipitates, the group treated orally administered to mice with SDS was treated with SDS treated TX, and the group treated orally treated with ProK treated with mice was treated with ProK treated TX. The number of acid spheres was calculated (FIG. 14). As a result, the eosinophil count was significantly decreased in the ProK-treated TX group compared to the TX group in both cases of 3 days and 5 days after administration, and a significant difference was observed. In the SDS-treated TX group, a slight but significant decrease was observed.

[Discussion]
In Example 3 of the present invention, in order to verify whether Th2 immunostimulation occurs by oral administration of the Nb-derived desurfactant fraction, various reactions used as indicators of Th2 immunostimulation were used. . As a result, a marked increase in the number of eosinophils in peripheral blood and the amount of IL-4 mRNA in mesenteric lymph nodes was observed 7 days after administration (FIGS. 5 and 7). Furthermore, the number of eosinophils in peripheral blood in the AW group to which all the components of Nb were administered was significantly increased as compared with the PBS group to which PBS was administered, but the ratio was administered to the desurfactant fraction. Compared with TX group, it was significantly lower. This not only indicates that the active ingredient for Th2 immunostimulation is contained in Nb, but also that the active ingredient is mainly contained in the desurfactant fraction separated by this example. Show.

  An increase in the amount of IL-4 mRNA and an increase in the number of eosinophils in the mesenteric lymph node was observed 7 days after administration, but an increase in IgE concentration in peripheral blood was not observed 7 days after administration. Recognized in 14 days. On the other hand, the number of eosinophils decreased to the same extent as in the PBS group 14 days after administration. These results indicate that administration of the Nb-derived desurfactant fraction causes Th2 immunostimulation to occur at least on day 7 and Th2 immunostimulation levels return to normal on day 14, Activation of the Th2 immune response increases IL-4 production, resulting in increased IgE concentrations. Previously, the inventor has shown that Nb is almost eliminated by 7 days after administration, Th2 immunostimulation is essential for its elimination, and that an increase in IgE concentration is first observed 14 days after administration. The present example supports these results.

Known biological reactions observed during Nb infection include increased mast cells and goblet cells in the intestinal tract, increased mucus secretion from goblet cells, and swelling of mesenteric lymph nodes and Peyer's patches (for example, non-patented) Reference 17). In the small intestine infected with Nb, edema occurs due to leukotriene and heparin produced by activated mucosal mast cells (see, for example, Non-Patent Document 9), increased permeability of the intestinal epithelium by release of histamine, and the like (for example, Non-Patent Document 9). It is reported that the increase in the number of mast cells is also caused by the increase in production of Th2 cytokine (for example, see Non-Patent Document 11 and Non-Patent Document 17). Even in the administration using the Nb-derived desurfactant fraction in this example, edema due to Th2 immunostimulation occurred, and as a result, the small intestine weight of the TX group was expected to increase. There were few results (FIG. 2). Furthermore, from the histopathological observation, no reactive changes such as inflammation were observed in the small intestine of the TX group, and the number of mast cells and goblet cells was not significantly increased compared to the PBS group (Fig. 8 to 13). In addition to Th2 cytokines, it has been reported that growth factors such as SCF (Stem Cell Factor) are also involved in the increase in the number of mast cells (see, for example, Non-Patent Document 17). In addition, in mutant (W / W ^v ) mice with SCF receptor dysfunction, it has been shown that the number of mast cells does not increase even when increased production of Th2 cytokine occurs during gastrointestinal parasite infection (for example, non-patented) Reference 15). From these prior art findings, in Example 3, the number of mast cells and the number of goblet cells increased in spite of the increase in IL-4 production in the lymph nodes and the increase in the number of eosinophils. As a cause of the absence, it is considered that administration of the Nb-derived desurfactant fraction did not affect the production of SCF or the like unlike the parasitic infection. Further, since there is a report that IL-13, which is one of Th2 cytokines, is essential for the proliferation of goblet cells (see, for example, Non-Patent Document 10), the reason why the number of goblet cells has not increased is described in more detail. Therefore, it is necessary to detect the production amount of IL-13. Furthermore, Th2 immunostimulation by gastrointestinal parasites is different in the level of activation depending on the type of parasite (see, for example, Non-Patent Document 17), differentiation and proliferation of eosinophils, production of IgE, It has also been reported that there are differences in Th2 cytokines involved in the increase in the number of goblet cells, mast cells, etc. (see, for example, Non-Patent Document 17). In order to analyze in detail, it is considered necessary to verify based on changes in the amount of each cytokine produced.

  The activation mechanism of the Th2 immune reaction is that, after an antigen is taken up by an antigen-presenting cell, the antigen-treated peptide fragment binds to an MHC molecule by processing, and then the antigen expressed on the cell surface of the antigen-presenting cell is expressed by T A mechanism in which Th2 immunostimulation occurs when a cell recognizes and sensitizes, and then an antigen enters the T cell, and TLR2 present on the cell surface of the antigen-presenting cell (for example, Non-Patent Document 3, Non-Patent Document) 14) and the like, the mechanism of causing Th2 immunostimulation can be mentioned, but the prior art known to cause Th2 immunostimulation by frequent administration mixed with an adjuvant is known. (For example, see Non-Patent Document 6 and Non-Patent Document 12), which is considered to be due to the former mechanism. It is given by, and a significantly Th2 immune response in the administration of single dose since that could be activated, can be attributed to the more direct latter mechanism. The discovery of substances that directly act on antigen-presenting cells and induce Th1 immunostimulation such as LPS and CpG derived from bacteria has advanced research on the initial reaction of the Th1 immunostimulation mechanism (for example, Non-Patent Document 4, Non-Patent Documents). From Reference 14), if it is found that the Nb-derived desurfactant fraction used in the present invention directly acts on antigen-presenting cells, the mechanism of the initial reaction of the Th2 immunostimulatory mechanism still has many unclear points. It is thought that it leads to elucidation of.

  It is considered that various immune diseases occur when the balance of the Th1 / Th2 immune response is lost. Examples of diseases in which the Th1 / Th2 immune reaction balance is excessively inclined toward Th1, that is, Th1 diseases include Crohn's disease, rheumatoid arthritis, autoimmune hepatitis and the like. Among these, Crohn's disease is called inflammatory bowel disease together with ulcerative colitis, and is known to be a chronic disease that can develop in the entire digestive tract. Although the cause is still unknown, it has a genetic predisposition and is thought to cause symptoms such as inflammation mainly due to mucosal damage of IFN-γ, and to develop by overactivating the Th1 immune response (for example, (See Non-Patent Document 7 and Non-Patent Document 13). There is no fundamental treatment, and remission is achieved with antiseptic analgesics such as aminosalicylic acid preparations and steroids, and immunosuppressants have been used to treat Crohn's disease in severe cases. However, serious side effects are concerned with steroids and immunosuppressants, and in recent years, infliximab, an anti-TNF-α antibody preparation, is commonly used, but it is difficult to maintain long-term remission with a single administration Therefore, it is necessary to administer continuously (for example, see Non-Patent Document 13). Therefore, although the active component having strong Th2 immunostimulatory activity is likely to be a peptide (including protein) in the desurfactant fraction of the present invention (FIG. 14), identification of such active component and molecular structure If further research such as elucidation proceeds, it is expected to lead to the development of new treatment methods such as Crohn's disease that is excessively inclined to Th1 type.

  Since the composition having Th2 immunostimulatory activity in the present invention can induce very strong Th2 immunostimulatory by a single oral administration, by conducting research such as identification of its active ingredients and elucidation of its molecular structure It can be expected to lead to the development of drugs and vaccines as Th2 immunostimulators.

Claims (9)

Nippo stolons di Las brasiliensis down cis Th2 immune characterized by (Nippostrongylus brasiliensis) consist solely de-detergent extraction fraction surfactant has been removed from the detergent fraction extracted using detergent from Activator (except food). The Th2 immunostimulator according to claim 1, wherein the desurfactant-extracted fraction contains a peptide. The Th2 immunostimulator according to claim 1 or 2, wherein the surfactant is TritonX-114 (registered trademark). The Th2 immunostimulator according to any one of claims 1 to 3 , which is used in a form of oral administration. A pharmaceutically acceptable normal carrier, binder, stabilizer, excipient, diluent, pH buffer, disintegrant, and solubilizer for the Th2 immunostimulator according to any one of claims 1 to 4. , A composition for Th2 immunostimulation to which a solubilizing agent or an isotonic agent is added. The method for producing a Th2 immunostimulator according to claim 1, comprising the following steps (a) to (d).
(A) a step of mixing Nippostrongylus brasiliensis or a treated product thereof with a surfactant-containing aqueous solution;
(B) a step of performing a layer separation treatment between the aqueous layer and the surfactant layer;
(C) collecting a surfactant fraction;
(D) a step of removing the surfactant from the surfactant fraction to obtain only the desurfactant extracted fraction; 7. The production method according to claim 6 , wherein the treated product of Nippostrongillus braziliansis is a physically destroyed treated product of Nippostrongillus braziliansis. The production method according to claim 6 or 7 , wherein the surfactant is TritonX-114 (registered trademark). The production method according to any one of claims 6 to 8 , wherein the surfactant removal treatment in the step (d) includes a protein precipitation treatment.