WO1995028180A1 - Simple method for testing immune function - Google Patents
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- WO1995028180A1 WO1995028180A1 PCT/JP1995/000744 JP9500744W WO9528180A1 WO 1995028180 A1 WO1995028180 A1 WO 1995028180A1 JP 9500744 W JP9500744 W JP 9500744W WO 9528180 A1 WO9528180 A1 WO 9528180A1
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- 230000036737 immune function Effects 0.000 title claims abstract description 8
- 238000012360 testing method Methods 0.000 title claims description 16
- 238000000034 method Methods 0.000 title abstract description 4
- 241001465754 Metazoa Species 0.000 claims abstract description 18
- 238000010171 animal model Methods 0.000 claims abstract description 17
- 239000000126 substance Substances 0.000 claims abstract description 16
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 10
- 108090000695 Cytokines Proteins 0.000 claims abstract description 9
- 102000004127 Cytokines Human genes 0.000 claims abstract description 9
- 210000002966 serum Anatomy 0.000 claims abstract description 9
- 230000016396 cytokine production Effects 0.000 claims abstract description 6
- 230000000694 effects Effects 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 238000010998 test method Methods 0.000 claims description 9
- 108010002616 Interleukin-5 Proteins 0.000 claims description 8
- 108090000978 Interleukin-4 Proteins 0.000 claims description 7
- 102000004388 Interleukin-4 Human genes 0.000 claims description 7
- 108010002350 Interleukin-2 Proteins 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 4
- 102000004856 Lectins Human genes 0.000 claims description 3
- 108090001090 Lectins Proteins 0.000 claims description 3
- 230000032823 cell division Effects 0.000 claims description 3
- 238000001990 intravenous administration Methods 0.000 claims description 3
- 239000002523 lectin Substances 0.000 claims description 3
- 230000005965 immune activity Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 230000003013 cytotoxicity Effects 0.000 claims 1
- 231100000135 cytotoxicity Toxicity 0.000 claims 1
- 238000011002 quantification Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 12
- 201000011510 cancer Diseases 0.000 abstract description 11
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 5
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 3
- 238000011156 evaluation Methods 0.000 abstract description 3
- 230000003266 anti-allergic effect Effects 0.000 abstract description 2
- 239000000411 inducer Substances 0.000 abstract description 2
- 230000002434 immunopotentiative effect Effects 0.000 abstract 1
- 230000003449 preventive effect Effects 0.000 abstract 1
- 241000186427 Cutibacterium acnes Species 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 241000244186 Ascaris Species 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000885184 Scaris Species 0.000 description 5
- 230000001093 anti-cancer Effects 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 238000002054 transplantation Methods 0.000 description 5
- 208000035143 Bacterial infection Diseases 0.000 description 4
- 208000022362 bacterial infectious disease Diseases 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
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- 238000001727 in vivo Methods 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 239000000043 antiallergic agent Substances 0.000 description 2
- 230000005907 cancer growth Effects 0.000 description 2
- 210000002443 helper t lymphocyte Anatomy 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 239000003226 mitogen Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 208000020154 Acnes Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
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- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 206010061259 Klebsiella infection Diseases 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
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- 238000003776 cleavage reaction Methods 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
- 230000002548 cytokinetic effect Effects 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
Definitions
- the present invention is directed to a serum cytokinin (IL-2, IL-4, IL-5, IFN-5) induced by intravenous administration of a T cell mitogen to a test animal pre-administered with a test substance. Quantify the amount of production, and calculate the production ratio of IFN-rZIL-4, IFN- ⁇ / ⁇ L-5, IL-2ZIL-4 or IL-2ZIL-5.
- the present invention makes it possible to evaluate the immune function of each individual animal, to prevent infection by immunostimulation and to treat cancer, and to a simple test method for anti-inflammatory and anti-allergic agents. Background art
- IL-2 was secreted into the blood of animals at the onset of ConA-induced liver injury (Journal of Clinical Investige). Icyon, Vol. 90, pp. 196, 1992). Therefore, we examined the possibility that other cytokines might be secreted into the blood by administering C0nA-like mitogens of immunocompetent cells to experimental animals, and as a result of investigations based on the prediction, we found that It was found that many cytokines including the above-mentioned cytokines were secreted, and based on this finding, the present invention was completed.
- the present invention quantifies the concentration of cytokines in serum induced by intravenous administration of a T cell cleavage inducer after single or continuous administration of a test substance to an experimental animal, and This is a simple test method characterized by evaluating the immune function of an individual animal.
- the T cell division inducing substance is concanapalin A (ConA), phytohemagglutin (PHA), or wheatgram lectin (WGL).
- the experimental animal is preferably a mouse or a rat, and the production rate of serum cytoin of the animal to which the solvent used at the time of administration of the test substance was administered (IFN-a / IL-4, IFN- 7 / IL-5, IL-1 2 IL-4 or IL-2ZIL-5).
- Henoleper T cells produce various cytokines when activated by the antigen ⁇ lectin.
- Helper T cells are classified into two subsets, Thl and Th2, depending on the type of cytoplasmic force produced in the mouse.
- Thl produces IL-2, IFN- ⁇ and INF-yS
- Th2 produces IL-14, IL-15, IL-6 and IL-10 and examines their production ratio This makes it possible to evaluate which of the D-111 and D- 12 Helme-1 T cell functions are dominant.
- helper T cells When T cells are activated in vivo, the interaction of helper T cells and sublesser T cells may be considered due to the operation of a complex cytoile network, and there is a considerable difference between the in vitro results and the immunoreactivity of individual animals.
- the present invention is characterized in that a test substance is administered to an experimental animal, allowed to stand for a certain period of time in order to distribute the test substance in the body, and then a certain dose of a T cell differentiation-inducing substance (for example, ConA) is intravenously administered. Administer.
- a T cell differentiation-inducing substance for example, ConA
- the serum is diluted appropriately, the above cytokine is quantified by enzyme immunoassay, and its production ratio (IFN-a ZIL-4, IFN-a ZIL-5, IL-2 / IL-4 or IL-4 -2ZI L-5, etc.), the solvent-administered group and the test substance-administered group are compared, and the smaller the value is compared to the control group, the stronger the infection protection, anti-cancer action, and anti-viral effect It shows anti-allergic and anti-inflammatory effects as the value increases.
- Fig. 1 Effects of P. acnes and Ascaris-treated animals on Klebsiella pneumoniae infection.
- Fig. 2 Effects of P. acnes and Ascaris-treated animals on Lewis lung cancer.
- mice As experimental animals, 5 to 10 male C57BL / 6 mice weighing about 25 g were used per group.
- the tumor mass of Lewis lung carcinoma subcultured by subcutaneous administration to a C57B LZ6 male mouse is excised, washed in a Petri dish containing Hanks' solution, and the tissue is finely separated with scissors. Thereafter, the cells were cultured in a nutrient solution containing 0.2% collagenase for 1 hour. After separating the cells, the survival rate was measured using a trypan, and the cells were adjusted to 5 ⁇ 10 5 cancer cells.
- an untreated group were set up, and each group was left for one week after the final administration.
- the cancer cells obtained by the above operation were subcutaneously administered at a ratio of 10 5 ce 11 sZ mice. The survival rate was observed for 40 days after the treatment.
- the control group gradually died from the 18th day after transplantation of the cancer cells, and 50% died on the 30th day and 70% on the 40th day from experimental animal cancer.
- deaths were observed on day 17 after cancer cell transplantation, and 70 on day 30. 80 in 40 days? Of the experimental animals died, and mortality was always ahead of the control group.
- deaths occurred 23 days after the transplantation of cancer cells, but only 30% of the animals died 40 days after the transplantation.
- Table 2 Relationship between IFN-r / IL-5 and antibacterial and anticancer effects Treatment group / IL-5 antibacterial activity * Anticancer activity # vs. 1 group 1 1. 4 10 50
- IFN-rZIL-5 becomes larger than that of the control group, it is not clear about bacterial infection, but cancer growth is promoted. Further, it was considered that when the values became smaller, these effects were suppressed and acted to prevent bacterial infection and suppress cancer growth.
- the value of IFN-rZIL-5 is a value that can reflect the immune function of an experimental animal individual. Using this value, it is a highly useful test model that can easily evaluate the immune activity of a test substance in vivo.
- test method of the present invention prevention of infection by immunostimulation, treatment of cancer, anti-inflammation, and in vitro evaluation of an antiallergic agent can be easily performed.
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Abstract
A simple method for evaluating the immune function of an individual experimental animal according to the cytokine production pattern obtained by administering a sample substance to the animal once or repeatedly, then intravenously administering a T-cell division inducer, and determining the serum cytokine level realized thereby. This method facilitates in vitro evaluation of infection preventive, cancer remedy, anti-inflammatory and anti-allergic by immunopotentiation.
Description
明細書 免疫機能の簡易試験法 技術分野 Description Simple test method for immune function
本発明は被検物質を前投与した実験動物に T細胞分裂誘導物質を静脈内投与す ることにより惹起される、 血清中サイト力イン （ I L— 2, I L-4, I L— 5, I F N—ァ等） 産生量を定量し、 特に I FN— rZI L— 4， I FN- γ/Ι L -5, I L— 2ZI L— 4もしくは I L— 2ZI L— 5の産生比率を算出するこ とにより、 動物個体別の免疫機能を評価でき、 免疫賦活による感染防止および癌 治療、 抗炎症、 抗アレルギー剤の簡易試験法に関する。 背景技術 The present invention is directed to a serum cytokinin (IL-2, IL-4, IL-5, IFN-5) induced by intravenous administration of a T cell mitogen to a test animal pre-administered with a test substance. Quantify the amount of production, and calculate the production ratio of IFN-rZIL-4, IFN-γ / ΙL-5, IL-2ZIL-4 or IL-2ZIL-5. The present invention makes it possible to evaluate the immune function of each individual animal, to prevent infection by immunostimulation and to treat cancer, and to a simple test method for anti-inflammatory and anti-allergic agents. Background art
動物個体の免疫機能を評価する試験法はこれまでになく、 全て in vitroの試験 (ジャーナル ·オフ、' ·ィムノロジ一， 第 136巻、 第 2348頁、 1986年） で評価が行われていた。 また従来の方法による評価では生体の複雑なサイトカイ ンネットワークの影響が考慮されておらず、 in vitro と in vivo の試験結果に は大きな差異もみられた。 発明の開示 There has never been a test method to evaluate the immune function of an animal individual, and all of the methods have been evaluated in in vitro tests (Journal Off, 'Immunology I, Vol. 136, p. 2348, 1986). In addition, the evaluation by the conventional method did not take into account the effects of the complex cytokinetic network of the living body, and there were significant differences between in vitro and in vivo test results. Disclosure of the invention
この様な問題点を解決するために本発明者らは、 まず C o n A誘発肝障害発症 時に I L— 2が動物の血中に分泌されている （ジャーナル ·ォブ ·クリニカル · ィンべスティゲ一シヨン、 第 90巻、 第 1 96頁、 1 992年） ことに注目した。 そこで C 0 n A様の免疫担当細胞の分裂誘導物質を実験動物に投与することに より、 血中にその他のサイトカインも分泌されるのではないかと予測にもとずき 検討した結果、 実際に上記サイトカインを含め多くのサイトカインが分泌されて いることを見し、だし、 この知見に基ずき本発明を完成させた。 In order to solve such a problem, the present inventors first found that IL-2 was secreted into the blood of animals at the onset of ConA-induced liver injury (Journal of Clinical Investige). Icyon, Vol. 90, pp. 196, 1992). Therefore, we examined the possibility that other cytokines might be secreted into the blood by administering C0nA-like mitogens of immunocompetent cells to experimental animals, and as a result of investigations based on the prediction, we found that It was found that many cytokines including the above-mentioned cytokines were secreted, and based on this finding, the present invention was completed.
すなわち本発明は、 実験動物に被検物質を単回もしくは連続投与後、 T細胞分 裂誘導物質を静脈内投与することにより惹起される血清中のサイトカイン濃度を 定量し、 サイト力インの産生パターンにより、 その動物個体の免疫機一能を評価す ることを特長とする簡易試験法である。
本発明において、 T細胞分裂誘導物質がコンカナパリ ン A (C o n A) 、 フィ 卜へマグルチン （PHA) またはウイ一トグラムレクチン （WGL) であること が好ましい。 That is, the present invention quantifies the concentration of cytokines in serum induced by intravenous administration of a T cell cleavage inducer after single or continuous administration of a test substance to an experimental animal, and This is a simple test method characterized by evaluating the immune function of an individual animal. In the present invention, it is preferable that the T cell division inducing substance is concanapalin A (ConA), phytohemagglutin (PHA), or wheatgram lectin (WGL).
また、 実験動物がマウスまたはラットであることが好ましく、 被検物質の投与 時に使用した溶媒を投与した動物の血清中サイ卜力イン産生比率 （I FN—ァ/ I L- 4, I FN- 7/ I L- 5, I L一 2ノ I L— 4もしくは I L— 2ZI L 一 5) と比較することが好ましい。 The experimental animal is preferably a mouse or a rat, and the production rate of serum cytoin of the animal to which the solvent used at the time of administration of the test substance was administered (IFN-a / IL-4, IFN- 7 / IL-5, IL-1 2 IL-4 or IL-2ZIL-5).
へノレパー T細胞は抗原ゃレクチンで活性化されると種々のサイトカインを産生 する。 マウスにおいて産生されるサイ卜力インの種類によって、 ヘルパー T細胞 は Thlと Th2の 2つのサブセットに分類される。 Thlは I L— 2、 I FN- γ および I NF— ySを産生するのに対し、 Th2は I L一 4、 I L一 5、 I L— 6お よび I L— 10を産生し、 その産生比率を検討することにより、 丁111と丁12の どちらのヘルメ 一 T細胞機能が優位になつているのか評価できる。 Henoleper T cells produce various cytokines when activated by the antigen ゃ lectin. Helper T cells are classified into two subsets, Thl and Th2, depending on the type of cytoplasmic force produced in the mouse. Thl produces IL-2, IFN-γ and INF-yS, whereas Th2 produces IL-14, IL-15, IL-6 and IL-10 and examines their production ratio This makes it possible to evaluate which of the D-111 and D- 12 Helme-1 T cell functions are dominant.
インビボで T細胞を活性化させると複雑なサイトカイレネットワークの作動に よりヘルパー T細胞とサブレッサ一 T細胞との相互作用も考えられ、 インビトロ の結果と動物固体の免疫活性にはかなりの差異が見られる。 When T cells are activated in vivo, the interaction of helper T cells and sublesser T cells may be considered due to the operation of a complex cytoile network, and there is a considerable difference between the in vitro results and the immunoreactivity of individual animals. Can be
本発明を詳述すれば、 実験動物に被検物質を投与し、 体内に分布させるため一 定時間の放置させた後、 T細胞分化誘導物質 （例えば C o n A) の一定用量を静 脈内投与する。 In detail, the present invention is characterized in that a test substance is administered to an experimental animal, allowed to stand for a certain period of time in order to distribute the test substance in the body, and then a certain dose of a T cell differentiation-inducing substance (for example, ConA) is intravenously administered. Administer.
一定時間放置後、 麻酔下に採血し遠心分離 （3000 r pm、 15分） によ り血清を採取する。 After leaving for a certain period of time, collect blood under anesthesia, and collect serum by centrifugation (3000 rpm, 15 minutes).
この血清について適度の希釈を行い酵素免疫法により上記サイトカインを定量 し、 その産生比率 （ I FN—ァ ZI L— 4, I FN—ァ ZI L— 5, I L- 2/ I L— 4もしくは I L— 2ZI L— 5など） について、 溶媒投与群と被検物質投 与群を比較し、 その値が対照群の値に比較し小さくなるほど、 感染防御、 抗癌作 用、 抗ウィルス作用が強く、 値カ大きくなるにつれて抗アレルギー作用および抗 炎症作用を示すものである。 図面の簡単な説明 The serum is diluted appropriately, the above cytokine is quantified by enzyme immunoassay, and its production ratio (IFN-a ZIL-4, IFN-a ZIL-5, IL-2 / IL-4 or IL-4 -2ZI L-5, etc.), the solvent-administered group and the test substance-administered group are compared, and the smaller the value is compared to the control group, the stronger the infection protection, anti-cancer action, and anti-viral effect It shows anti-allergic and anti-inflammatory effects as the value increases. BRIEF DESCRIPTION OF THE FIGURES
図 1 P. acnes および Ascaris 処置後動物の肺炎桿菌感染に対する作用 図 2 P. acnes および Ascaris処置後動物のルイス肺癌に対する作用
発明を実施するための最良の形態 Fig. 1 Effects of P. acnes and Ascaris-treated animals on Klebsiella pneumoniae infection. Fig. 2 Effects of P. acnes and Ascaris-treated animals on Lewis lung cancer. BEST MODE FOR CARRYING OUT THE INVENTION
以下に試験例を挙げて本発明を具体的に説明する。 Hereinafter, the present invention will be specifically described with reference to test examples.
実験動物は、 体重約 25 gの C 57BL/6系雄性マウスを 1群 5 _ 10匹で 使用した。 As experimental animals, 5 to 10 male C57BL / 6 mice weighing about 25 g were used per group.
実施例 1 [サイト力イン産生に対する作用] Example 1 [Effect on Site Force In Production]
上記実験動物にァスカリス抽出エキス （A s c a r i s) を蛋白量にして 2m gZk gを 1週間毎に 1回、 合計 3回皮下投与する群。 またプロピオバクテリウ ム ·ァクネス （P. a c n e s) の加熱死菌 5mgZk gを静脈内投与する群。 および無処置群の 3群を設け、 各群とも最終投与後 1週間放置した実験動物につ いて、 C o nA5mgZk gを静脈内 Π投与した。 4時間の放置後血清中の I F N—ァおよび I L— 5濃度を定量し、 その比率 （I FN/I L-5) を算出した。 (実験結果） A group in which the above experimental animals were subcutaneously administered with 2 mg / kg of the Ascaris extract (A scaris) once a week for a total of three times. In addition, a group to which 5 mgZkg of heat-killed Propiobacterium acnes (P. acnes) was administered intravenously. And an untreated group, and each group was administered intravenously with 5 mg of ConA 5 mgZkg for the experimental animals left for one week after the final administration. After standing for 4 hours, the concentration of IFN-I and IL-5 in the serum was quantified, and the ratio (IFN / IL-5) was calculated. (Experimental result)
表 1 P. acnes および Ascaris処置後の血中サイトカイン産生量とその比率 Table 1 Blood cytokine production after treatment with P. acnes and Ascaris and their ratio
表 1に示す様に、 実験動物にある種の前処置を行うことによりサイトカイン産 生はその種類により大きく影響を受ける。 As shown in Table 1, certain types of pretreatment of experimental animals significantly affect cytokine production.
例えば P. a c n e s処置群では I F N— 7産生の著しい増加を促し、 逆に A s c a r i s処置群では I L— 5の産生量を増加させるため、 上記 2種のサイ卜 カイン産生比率を算出すると。 対照群の値に比し逆の変動がみられることがわか る。 次にこの様な状態時に、 細菌感染および癌移植にどの様な影響がみられるの か検討した。 実施例 2 [細菌感染に対する作用] For example, when the P. acnes treatment group promotes a marked increase in IFN- 7 production, and conversely, the A scaris treatment group increases the amount of IL-5 production. It can be seen that the change is opposite to that of the control group. Next, we examined the effects of these conditions on bacterial infection and cancer transplantation. Example 2 [Action on bacterial infection]
上記の実験動物にァスカリス抽出エキス （A s c a r i s) を蛋白量にして 2 mg/k gを 1週間毎に 1回、 合計 3回皮下投与する群。 またプロピオバクテリ
ゥム .ァクネス （P. a c n e s) の加熱死菌 5mgZk gを静脈内投与する群。 および無処置群の 3群を設け、 各群とも最終投与後 1週間放置した実験動物につ いて、 5%ムチン溶液に K. p n e umo n i a e 6 (肺炎桿菌） 2. 44X 1 07CFU/マウスを 0. 5m 1 /マウスの割合で腹腔内投与した。 その後 7日間 の動物の生存率を観察して感染予防効果を評価した。 A group in which 2 mg / kg of Ascaris extract extract (A scaris) is administered subcutaneously to the above experimental animals once a week for a total of three times. Propobacteria A group to which 5 mgZkg of heat-killed P. acnes was administered intravenously. And three groups of untreated group provided, with each group had experimental animals Nitsu allowed to stand for 1 week after the last dose, K. pne umo niae 6 (Klebsiella pneumoniae) in 5% mucin solution 2. 44X 1 0 7 CFU / mouse Was intraperitoneally administered at a rate of 0.5 ml / mouse. After that, the survival rate of the animals for 7 days was observed to evaluate the infection prevention effect.
(実験結果） (Experimental result)
図 1に示した様に、 対照群の実験動物では肺炎桿菌処置後 24時間以内に 9 0%の動物力、'死亡したのに対し、 P. a c n e s処置群では有意な変化はみられ なかった。 しかし A s c a r i s処置群においては肺炎桿菌処置後 48時間以内 に 50%の実験動物が死亡後 7日目まで、 死亡例は観察されなかった。 以上の結 果より、 A s c a r i s処置群においては感染抑制作用があるものと考えられた。 実施例 3 [抗癌作用] As shown in Figure 1, the control animals died 90% of the animal power within 24 hours after K. pneumoniae treatment, while the P. acnes-treated group did not show significant changes. . However, in the Ascaris-treated group, no deaths were observed until 7 days after the death of 50% of the experimental animals within 48 hours after K. pneumoniae treatment. From the above results, it was considered that the Ascaris-treated group had an effect of suppressing infection. Example 3 [Anti-cancer action]
C 57 B LZ6系雄性マウスに皮下投与して継代した L e w i s肺癌腫瘍塊 を 13日目に摘出し、 ハンクス液の入ったシャーレ中で洗浄後、 組織をハサミで 細かく分離する。 その後 0. 2%コラゲナ一ゼの入った栄養液中で 1時間培養し、 細胞を分離後、 トリパンブル一で生存率を計測し、 癌細胞 5 X 105ノ m 1に調製 した。 On the 13th day, the tumor mass of Lewis lung carcinoma subcultured by subcutaneous administration to a C57B LZ6 male mouse is excised, washed in a Petri dish containing Hanks' solution, and the tissue is finely separated with scissors. Thereafter, the cells were cultured in a nutrient solution containing 0.2% collagenase for 1 hour. After separating the cells, the survival rate was measured using a trypan, and the cells were adjusted to 5 × 10 5 cancer cells.
C 57 B LZ 6系雄性マウスにァスカリス抽出エキス （A s c a r i s ) を蛋 白量にして 2mgZk gを 1週間毎に 1回、 合計 3回皮下投与する群。 またプロ ピオバクテリゥム ·ァクネス （P. a c n e s) の加熱死菌 5mg/k gを静脈 内投与する群。 および無処置群の 3群を設け、 各群とも最終投与後 1週間放置し -た動物について、 上記操作によって得られた癌細胞を 105c e 1 1 sZマウスの 割合で皮下投与し、 癌細胞処置後 40日間の生存率を観察した。 A group of subcutaneous administration of 2 mg Zkg once a week to the C57B LZ6 male mice, using ascaris extract extract (A scaris) as a protein amount, once a week for a total of three times. In addition, a group receiving 5 mg / kg of heat-killed P. acnes bacteria administered intravenously. And an untreated group were set up, and each group was left for one week after the final administration.- For the animals, the cancer cells obtained by the above operation were subcutaneously administered at a ratio of 10 5 ce 11 sZ mice. The survival rate was observed for 40 days after the treatment.
(実験結果） (Experimental result)
図 2に示した様に、 対照群では癌細胞移植後 18日目より徐々に死亡例が認め られ、 30日目では 50%、 40日目では 70%の実験動物力癌により死亡した。 P. a c n e s処置群では、 癌細胞移植後 17日目より死亡例が認められ、 30 日目では 70？ 、 40日では 80？ の実験動物が死亡し、 対照群に比し死亡率が 常に先行した。 A s c a r i s処置群においては、 癌細胞移植後 23日目より死 亡例が認められたが 40日後においても 30%の動物しか死亡しなかった。
表 2 IFN-r/IL-5 と抗菌および抗癌作用の関係 処 群 / I L- 5 抗菌作用 * 抗癌作用 # 対 眧 群 1 1. 4 10 50As shown in Fig. 2, the control group gradually died from the 18th day after transplantation of the cancer cells, and 50% died on the 30th day and 70% on the 40th day from experimental animal cancer. In the P. acnes treatment group, deaths were observed on day 17 after cancer cell transplantation, and 70 on day 30. 80 in 40 days? Of the experimental animals died, and mortality was always ahead of the control group. In the A scaris-treated group, deaths occurred 23 days after the transplantation of cancer cells, but only 30% of the animals died 40 days after the transplantation. Table 2 Relationship between IFN-r / IL-5 and antibacterial and anticancer effects Treatment group / IL-5 antibacterial activity * Anticancer activity # vs. 1 group 1 1. 4 10 50
P. a c n e s処置群 267. 2 20 30P. a c n e s treatment group 267.2 20 30
A s c a r i s処置群 3. 9 50 80 A s c a ri s treatment group 3.9 50 80
*抗菌作用： 1週間生存率 （％) * Antibacterial activity: 1 week survival rate (%)
#抗癌作用： 30日間生存率 （％) # Anticancer activity: 30-day survival rate (%)
以上の結果より、 P. a c n e s処置群では癌の悪化が、 A s c a i s処置 群では癌の抑制が認められた。
From the above results, it was confirmed that cancer was worsened in the P. acnes treatment group and suppressed in the Ascais treatment group.
産業上の利用可能性 Industrial applicability
以上の結果をあわせて考えると、 IFN- rZIL-5 の数値が対照群に比し大きくな ると、 細菌感染については明確でなかったが、 癌増殖作用などは促進される。 ま た数値が小さくなると、 これらの作用は抑制され細菌感染防御および癌増殖抑制 作用的に作用すると考えられた。 IFN-rZIL-5 の数値は実験動物個体の免疫機能 を反映できる数値であり、 この数値を用いることにより被検物質の免疫活性を in vivoで簡便に評価出来る有用性の高い試験モデルである。 Considering the above results, if the value of IFN-rZIL-5 becomes larger than that of the control group, it is not clear about bacterial infection, but cancer growth is promoted. Further, it was considered that when the values became smaller, these effects were suppressed and acted to prevent bacterial infection and suppress cancer growth. The value of IFN-rZIL-5 is a value that can reflect the immune function of an experimental animal individual. Using this value, it is a highly useful test model that can easily evaluate the immune activity of a test substance in vivo.
本発明の試験法により、 免疫賦活による感染防止、 癌治療、 抗炎症、 および抗 ァレルギ一剤の in vitroによる評価が簡便に出来るようになつた。
By the test method of the present invention, prevention of infection by immunostimulation, treatment of cancer, anti-inflammation, and in vitro evaluation of an antiallergic agent can be easily performed.
Claims
1. 実験動物に被検物質を単回もしくは連続投与後、 τ細胞分裂誘導物質を静 脈内投与することにより惹起される血清中のサイトカイン濃度を定量し、 サイト 力インの産生ノ、。ターンにより、 その動物個体の免疫機能を評価することを特長と する簡易試験法。 1. Single or continuous administration of the test substance to the experimental animal, and then quantification of the cytokine concentration in the serum caused by intravenous administration of the τ cell division inducing substance, to produce cytoforce. A simple test method that evaluates the immune function of an individual animal by turns.
2. T細胞分裂誘導物質がコンカナパリン A (Co nA) 、 フィ 卜へマグルチ ン （PHA) またはウイ一トグラムレクチン （WGL) であることを特長とする 請求項 1記載のサイ卜カイン産生ミパターンに影響を及ぼす物質の簡易試験法。 2. The cytokine production pattern according to claim 1, wherein the T cell division inducing substance is concanapalin A (ConA), phytohemagglutin (PHA) or wheatgram lectin (WGL). A simple test method for substances that have an effect.
青 Blue
3. 実験動物がマウスまたはラットであることを特長とする請求項 1または請 求項 2記載のサイ卜カイン産生パターンに影響を及ぼす物質の簡易試験法。 3. The simple test method for a substance that affects a cytokine production pattern according to claim 1 or claim 2, wherein the experimental animal is a mouse or a rat.
4. 被検物質の投与時に使用した溶媒を投与した動物血清中のサイトカイン ( I L一 2, I L-4, I L一 5, I F N—ァ） 産生量のうち、 I FN— y/I L - 4, I FN- 7/I L- 5, I L一 2ZI L— 4もしくは I L— 2/I L— 5の比を算出することにより求められる、 サイトカイン産生パターンと被検物質 投与による同上のサイ卜力イン産生パターンを比較することにより、 動物個体に おける免疫活性状態を評価する簡易試験法。
4. Of the cytokine (IL-12, IL-4, IL-15, IFN-a) production in the serum of the animal to which the solvent used at the time of administration of the test substance was administered, I FN- y / IL-4 , IFN-7 / IL-5, IL-1ZIL-4 or IL-2 / IL-5 ratio, calculated by calculating the ratio of cytokine production pattern and cytotoxicity by the administration of a test substance. A simple test method that evaluates the immune activity status in animal individuals by comparing production patterns.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU22243/95A AU2224395A (en) | 1994-04-18 | 1995-04-17 | Simple method for testing immune function |
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| Application Number | Priority Date | Filing Date | Title |
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| JP6/78648 | 1994-04-18 | ||
| JP7864894 | 1994-04-18 |
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| WO1995028180A1 true WO1995028180A1 (en) | 1995-10-26 |
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| PCT/JP1995/000744 WO1995028180A1 (en) | 1994-04-18 | 1995-04-17 | Simple method for testing immune function |
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| WO (1) | WO1995028180A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989004665A2 (en) * | 1987-11-25 | 1989-06-01 | Cetus Corporation | Treatment of infections caused by a primary immunodeficiency with interleukin-2 |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989004665A2 (en) * | 1987-11-25 | 1989-06-01 | Cetus Corporation | Treatment of infections caused by a primary immunodeficiency with interleukin-2 |
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