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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist a
ab
ab
ac
Ryohei F. Tsuji , Masakazu Uramoto , Hiroyuki Koshino , Noriko M. Tsuji , Junji ad
ad
Magae , Kazuo Nagai
a
& Makari Yamasaki
a
Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113, Japan b
Division of Molecular Characterization, RIKEN (The Institute of Physical and Chemical Research), 2–1 Hirosawa, Wako-shi, Saitama, 351–01 Japan c
National Institute of Animal Industry PO Box 5, Norin-danchi, Tsukuba-shi, Ibaraki 305, Japan d
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 227, Japan Published online: 12 Jun 2014.
To cite this article: Ryohei F. Tsuji, Masakazu Uramoto, Hiroyuki Koshino, Noriko M. Tsuji, Junji Magae, Kazuo Nagai & Makari Yamasaki (1992) Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist, Bioscience, Biotechnology, and Biochemistry, 56:10, 1686-1689, DOI: 10.1271/bbb.56.1686 To link to this article: http://dx.doi.org/10.1271/bbb.56.1686
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (10), 1686-1689, 1992
Rapid Paper
Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist Ryohei F. TSUJI,t Masakazu URAMOTO,* Hiroyuki KOSHINO,* Noriko M. Tsun,** Junji MAGAE,*** Kazuo NAGAI,*** and Makari YAMASAKI Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan of Molecular Characterization, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan ** National Institute of Animal Industry PO Box 5, Norin-danchi, Tsukuba-shi, Ibaraki 305, Japan *** Department ofBioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 227, Japan Received May 18, 1992
Downloaded by [National Pingtung University of Science and Technology] at 04:10 30 December 2014
* Division
In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hindfootpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.
Recently, we established a novel screening method for immunomodulating substances which were effective in vivo. 1) Three kinds of antigen i.e. sheep red blood cells (SRBC) as a thymus dependent antigen and heat-killed Brucella abortus cells as a thymus independent antigen 2 ) for antibody production, and trinitrobenzene sulfonic acid (TNBS) for delayed type-hypersensitivity, were injected into identical mice. In our previous paper,l) we showed that functions of B cells, type 1 helper T cells (Th 1), type 2 helper T cells (Th2), and inflammatory cells could be separately evaluated in vivo in this system. We screened a selective immunomodulator among microbial metabolites and found that Streptomyces sp. Al502 (FERM P-12448) produced a DTH-specific suppressing compound which was identical with L-156,602 3 ) (Fig. 1). L-156,602 was found as a C5a receptor antagonist 3 ) and is a member of the recently discovered class of cyclic hexadepsipeptides such as azinothricin,4) A83586,5) variapeptin,6) and citropeptin. 7 ) In this paper, we report the unique in vivo immunomodulating properties of L-156,602 and also discuss the possibility of C5a-involvement in the efferent phase of DTH.
Materials and Methods Animals. Specific pathogen free CDP l' BALB/c, and C57BL/6 mice (female, 6-8 weeks old) were obtained from Charles River Japan Co., Ltd., Tokyo. Reagents. SRBC were purchased from Nippon Bio-Supp. Center (Tokyo). Heat-killed Brucella abortus cells were a kind gift from Dr K. t
Hirota (National Institute of Animal Health, Tsukuba-shi, Japan). To prepare TNP-SRBC, 20% SRBC solution was incubated with 0.02 mM TNBS for the DTH assay or 2 mM TNBS for antibody production in 5 ml of phosphate buffered saline (PBS), pH 7.4, for 30 min at room temperature. Resultant TNP-SRBC was then washed with PBS three times and resuspended in PBS. TNP-bovine serum albumin (BSA) was obtained by incubation of 4 mg/ml BSA with 2 mM TNBS in 5 ml of PBS for 30 min at 30°C. After dialysis against PBS, TNP-SRBC was lyophilized and stored. Absorbance at 405 nm of 2 mg/ml solution was 1.6. Peroxidase conjugated anti-mouse IgM + G antibody was a product of Kirkegaard & Perry Lab. Inc. (Maryland, U.S.A.).
H
HO
~ o oX H3C"'''''''
OH
H3C?:~
H
,/
NH
N/l1YHN.,'H :
I
H
o
C~
0
O
~H
CH3
NH
o
.. H
HO'N
.'
CH,
~/N'i----l O
HN
V
Fig. 1.
The Structure of L-156,602.
Present address: Noda Institute for Scientific Research, 399 Nada, Noda-shi, Chiba 278, Japan.
NII-Electronic Library Service
Immunomodulating Properties of L-156,602
Downloaded by [National Pingtung University of Science and Technology] at 04:10 30 December 2014
DTH assay. TNP-specific DTH was induced as described previously.!) In the case of TNP-SRBC specific DTH, 1 x 10 5 TNP-SRBC were iv injected into C57BL/6 mice. Five days later, DTH was elicited by sc injecting 1 x lOB TNP-SRBC in 40 pI into the left hind-footpad. In the case of evaluating efferent phase of DTH, 8 x 10 5 cells of 6.11 G in 40 pI of MEM medium which were collected 2 weeks after antigen stimulation with syngeneic spleen cells and 50 pg of {3-lactoglobulin were directly sc injected into the left hind-footpad. Twenty four hours later after the DTH elicitation, increase in footpad thickness ws evaluated with a dial caliper (Ozaki Mfg Co., Ltd., Tokyo). Antibody production assay. Evaluation of antibody production against SRBC and Brucella abortus was done as described previously.l.B) In the case of detecting the response to TNP-SRBC, 1 x 10 8 of TNP-SRBC was iv injected into C57BL/6 mice. Six days later, sera were obtained from these mice and the antibody titer against TNP was evaluated with an ELISA assay using TNP-BSA as the coating antigen. The wells of a microtiter plate were coated with 10 pg/ml TNP-BSA. After blocking wells with 1% BSA, sera which were serially diluted with PBS containing 1% BSA were added. Peroxidase conjugated anti-mouse IgM + G antibody diluted 1 : 200 in PBS containing 0.05% Tween-20 and 1% BSA was used as the second antibody. Peroxidase substrate system ABTS (2.2'-azino-di[3-ethylbenathiazoline sulfonate) (Kirkegaard & Perry Laboratories Inc.) was used for the enzyme reaction. Antibody titer was expressed as absorbance at 405nm.
1687
acetone. After removal of acetone, the extract was further extracted with ethyl acetate. The concentrated fraction of the extract was put on a silica gel column. After a thorough washing of the column with hexane, benzene, ethyl acetate and acetone, the column was eluted with acetone-methanol (1: 1). The active fractions were concentrated, and then put on a silica gel column which was eluted by chloroform-methanol (20: 1). The active fraction was chromatographed on HPLC with a Capcell Pak-C18 column. The active eluate with 70% acetonitrile was concentrated to dryness to yield a colorless powder (10 mg). In the positive and negative FAB mass spectra of the active compound, strong peaks were at m/z 863 (M + Na) +, 879 (M+K)+ and 839 (M-H)-, respectively. (HRFBMS m/z: (M + Na)+, Calcd. for C3sH64Ns013Na: 863.4491, found: 863.4496; (M-H)-, Calcd. for C3SH63Ng013: 839.4515, found: 839.4517). This suggested ,that the active compound was L-156,602. The lH_ and 13C-NMR and IR spectra of the compound were identical with those of L-156,602.
Results
Immunomodulating properties of L-156,602 When L-156,602 was ip injected into CDF1 mice which Taxonomy were immunized with SRBC, heat-killed Brucella abortus Cultural and physiological characterization was done cells, and TNBS, DTH against TNBS was selectively mainly according to the International Streptomyces Project 10 9 suppressed (Table I). This suppression was evident at doses (ISP) report ) and the methods described by Waksman. ) In the case of antibody production, titers over 0.25 mg/kg. Morphological, cultural, and physiological characteristics, were evaluated by agglutinating ability with or without and cell wall composition indicated that the culture was a 2-mercaptoethanol (2-Me) treatment. Since IgM is instrain of Streptomyces. activated by this treatment, 2-ME resistant titer mainly consists of IgG. In fact, in the case of antibody production Isolation and identification of active compound The medium for fermentation consisted of 2% glucose, against TI antigen, no 2-ME resistant antibody titer was 1% soluble starch, 0.1 % meat extract, 0.4% dry yeast, detected. By an in vivo treatment with L-156,602, antibody 2.50/0 soy bean meal, 0.2% NaCI, and 0.005% K 1 HP0 4. productions against both SRBC and Brucella abortus were The pH was adjusted to 7.3 before sterilization. The slightly enhanced. To assure this selectivity of L-156,602, DTH and antifermentation was done at 27°C for 5 days with agitation at 350 rpm. The resultant culture (40 liters) was filtered body production were induced against the same antigen, i.e., TNP-SRBC with the same immunizing route although and the mycelial cake was extracted with enough 80% Table I.
DTH-Specific Suppression by L-156,602 in an Identical Mouse Anti-SRBC Ab titer
Immunization
Anti-Brucella Ab titer
Dose
Increase in footpad thickness (cm -2)
-2ME
+2Me
-2Me
+2Me
0.0 0.0 0.1 0.25 0.5 1.0
N.D. a 4.8±0.4 5.3±0.6 5.5±0.0 5.2±0.3 5.8 ± 1.3
N.D. 3.7 ± 1.0 3.8±0.8 5.0±0.0 4.3±0.8 4.0±2.2
N.D. 4.0±0.7 4.7±0.6 4.7±0.6 4.0± 1.0 5.5 ±0.9
N.D. N.D. N.D. N.D. N.D. 1.0± 1.0
2.2±2.1b 0.3±0.3b
0.0 0.0 0.5
N.D. 6.0±0.0 6.7±0.6
N.D. 3.8±0.3 5.2±0.6
N.D. 3.8±0.6 4.3±0.3
N.D. N.D. N.D.
0.5±0.5 10.3 ±OA 4.0± LOb
Exp. 1 + + + + + Exp. 2 + +
1.3±OA 8.8± 1.0 8.5 ± 1.8 5.8±3.7
CDF 1 mice were iv immunized with 1 x 10 8 SRBC and 1 x 10 9 heat killed Brucella abortus cells on day O. On day 6, 1 x 109 heat killed Brucella abortus cells were secondarily iv injected. CDF 1 mice were also injected sc with 200 pI of 10mM TNBS on day 3. On day 11, 40 pI of 10mM TNBS was injected into the subcutaneous tissue of the footpads. Antibody titer was defined as agglutinating ability on day 12 and DTH was evaluated by increase in footpad thickness 24 hours after the DTH elicitation. Sera were pre-treated with 2-ME or PBS alone for 60 min at 37°C. L-156,602 was injected ip on 0,2,4,6,8 and 10 (Exp. 1) or 1,3,8 (Exp. 2). Data were shown as mean±SD. a N.D., not detected. b Statistically significant compared with immunized control mice (two tailed Studient's t-test p
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist a
ab
ab
ac
Ryohei F. Tsuji , Masakazu Uramoto , Hiroyuki Koshino , Noriko M. Tsuji , Junji ad
ad
Magae , Kazuo Nagai
a
& Makari Yamasaki
a
Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113, Japan b
Division of Molecular Characterization, RIKEN (The Institute of Physical and Chemical Research), 2–1 Hirosawa, Wako-shi, Saitama, 351–01 Japan c
National Institute of Animal Industry PO Box 5, Norin-danchi, Tsukuba-shi, Ibaraki 305, Japan d
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 227, Japan Published online: 12 Jun 2014.
To cite this article: Ryohei F. Tsuji, Masakazu Uramoto, Hiroyuki Koshino, Noriko M. Tsuji, Junji Magae, Kazuo Nagai & Makari Yamasaki (1992) Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist, Bioscience, Biotechnology, and Biochemistry, 56:10, 1686-1689, DOI: 10.1271/bbb.56.1686 To link to this article: http://dx.doi.org/10.1271/bbb.56.1686
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (10), 1686-1689, 1992
Rapid Paper
Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist Ryohei F. TSUJI,t Masakazu URAMOTO,* Hiroyuki KOSHINO,* Noriko M. Tsun,** Junji MAGAE,*** Kazuo NAGAI,*** and Makari YAMASAKI Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan of Molecular Characterization, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan ** National Institute of Animal Industry PO Box 5, Norin-danchi, Tsukuba-shi, Ibaraki 305, Japan *** Department ofBioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 227, Japan Received May 18, 1992
Downloaded by [National Pingtung University of Science and Technology] at 04:10 30 December 2014
* Division
In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hindfootpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.
Recently, we established a novel screening method for immunomodulating substances which were effective in vivo. 1) Three kinds of antigen i.e. sheep red blood cells (SRBC) as a thymus dependent antigen and heat-killed Brucella abortus cells as a thymus independent antigen 2 ) for antibody production, and trinitrobenzene sulfonic acid (TNBS) for delayed type-hypersensitivity, were injected into identical mice. In our previous paper,l) we showed that functions of B cells, type 1 helper T cells (Th 1), type 2 helper T cells (Th2), and inflammatory cells could be separately evaluated in vivo in this system. We screened a selective immunomodulator among microbial metabolites and found that Streptomyces sp. Al502 (FERM P-12448) produced a DTH-specific suppressing compound which was identical with L-156,602 3 ) (Fig. 1). L-156,602 was found as a C5a receptor antagonist 3 ) and is a member of the recently discovered class of cyclic hexadepsipeptides such as azinothricin,4) A83586,5) variapeptin,6) and citropeptin. 7 ) In this paper, we report the unique in vivo immunomodulating properties of L-156,602 and also discuss the possibility of C5a-involvement in the efferent phase of DTH.
Materials and Methods Animals. Specific pathogen free CDP l' BALB/c, and C57BL/6 mice (female, 6-8 weeks old) were obtained from Charles River Japan Co., Ltd., Tokyo. Reagents. SRBC were purchased from Nippon Bio-Supp. Center (Tokyo). Heat-killed Brucella abortus cells were a kind gift from Dr K. t
Hirota (National Institute of Animal Health, Tsukuba-shi, Japan). To prepare TNP-SRBC, 20% SRBC solution was incubated with 0.02 mM TNBS for the DTH assay or 2 mM TNBS for antibody production in 5 ml of phosphate buffered saline (PBS), pH 7.4, for 30 min at room temperature. Resultant TNP-SRBC was then washed with PBS three times and resuspended in PBS. TNP-bovine serum albumin (BSA) was obtained by incubation of 4 mg/ml BSA with 2 mM TNBS in 5 ml of PBS for 30 min at 30°C. After dialysis against PBS, TNP-SRBC was lyophilized and stored. Absorbance at 405 nm of 2 mg/ml solution was 1.6. Peroxidase conjugated anti-mouse IgM + G antibody was a product of Kirkegaard & Perry Lab. Inc. (Maryland, U.S.A.).
H
HO
~ o oX H3C"'''''''
OH
H3C?:~
H
,/
NH
N/l1YHN.,'H :
I
H
o
C~
0
O
~H
CH3
NH
o
.. H
HO'N
.'
CH,
~/N'i----l O
HN
V
Fig. 1.
The Structure of L-156,602.
Present address: Noda Institute for Scientific Research, 399 Nada, Noda-shi, Chiba 278, Japan.
NII-Electronic Library Service
Immunomodulating Properties of L-156,602
Downloaded by [National Pingtung University of Science and Technology] at 04:10 30 December 2014
DTH assay. TNP-specific DTH was induced as described previously.!) In the case of TNP-SRBC specific DTH, 1 x 10 5 TNP-SRBC were iv injected into C57BL/6 mice. Five days later, DTH was elicited by sc injecting 1 x lOB TNP-SRBC in 40 pI into the left hind-footpad. In the case of evaluating efferent phase of DTH, 8 x 10 5 cells of 6.11 G in 40 pI of MEM medium which were collected 2 weeks after antigen stimulation with syngeneic spleen cells and 50 pg of {3-lactoglobulin were directly sc injected into the left hind-footpad. Twenty four hours later after the DTH elicitation, increase in footpad thickness ws evaluated with a dial caliper (Ozaki Mfg Co., Ltd., Tokyo). Antibody production assay. Evaluation of antibody production against SRBC and Brucella abortus was done as described previously.l.B) In the case of detecting the response to TNP-SRBC, 1 x 10 8 of TNP-SRBC was iv injected into C57BL/6 mice. Six days later, sera were obtained from these mice and the antibody titer against TNP was evaluated with an ELISA assay using TNP-BSA as the coating antigen. The wells of a microtiter plate were coated with 10 pg/ml TNP-BSA. After blocking wells with 1% BSA, sera which were serially diluted with PBS containing 1% BSA were added. Peroxidase conjugated anti-mouse IgM + G antibody diluted 1 : 200 in PBS containing 0.05% Tween-20 and 1% BSA was used as the second antibody. Peroxidase substrate system ABTS (2.2'-azino-di[3-ethylbenathiazoline sulfonate) (Kirkegaard & Perry Laboratories Inc.) was used for the enzyme reaction. Antibody titer was expressed as absorbance at 405nm.
1687
acetone. After removal of acetone, the extract was further extracted with ethyl acetate. The concentrated fraction of the extract was put on a silica gel column. After a thorough washing of the column with hexane, benzene, ethyl acetate and acetone, the column was eluted with acetone-methanol (1: 1). The active fractions were concentrated, and then put on a silica gel column which was eluted by chloroform-methanol (20: 1). The active fraction was chromatographed on HPLC with a Capcell Pak-C18 column. The active eluate with 70% acetonitrile was concentrated to dryness to yield a colorless powder (10 mg). In the positive and negative FAB mass spectra of the active compound, strong peaks were at m/z 863 (M + Na) +, 879 (M+K)+ and 839 (M-H)-, respectively. (HRFBMS m/z: (M + Na)+, Calcd. for C3sH64Ns013Na: 863.4491, found: 863.4496; (M-H)-, Calcd. for C3SH63Ng013: 839.4515, found: 839.4517). This suggested ,that the active compound was L-156,602. The lH_ and 13C-NMR and IR spectra of the compound were identical with those of L-156,602.
Results
Immunomodulating properties of L-156,602 When L-156,602 was ip injected into CDF1 mice which Taxonomy were immunized with SRBC, heat-killed Brucella abortus Cultural and physiological characterization was done cells, and TNBS, DTH against TNBS was selectively mainly according to the International Streptomyces Project 10 9 suppressed (Table I). This suppression was evident at doses (ISP) report ) and the methods described by Waksman. ) In the case of antibody production, titers over 0.25 mg/kg. Morphological, cultural, and physiological characteristics, were evaluated by agglutinating ability with or without and cell wall composition indicated that the culture was a 2-mercaptoethanol (2-Me) treatment. Since IgM is instrain of Streptomyces. activated by this treatment, 2-ME resistant titer mainly consists of IgG. In fact, in the case of antibody production Isolation and identification of active compound The medium for fermentation consisted of 2% glucose, against TI antigen, no 2-ME resistant antibody titer was 1% soluble starch, 0.1 % meat extract, 0.4% dry yeast, detected. By an in vivo treatment with L-156,602, antibody 2.50/0 soy bean meal, 0.2% NaCI, and 0.005% K 1 HP0 4. productions against both SRBC and Brucella abortus were The pH was adjusted to 7.3 before sterilization. The slightly enhanced. To assure this selectivity of L-156,602, DTH and antifermentation was done at 27°C for 5 days with agitation at 350 rpm. The resultant culture (40 liters) was filtered body production were induced against the same antigen, i.e., TNP-SRBC with the same immunizing route although and the mycelial cake was extracted with enough 80% Table I.
DTH-Specific Suppression by L-156,602 in an Identical Mouse Anti-SRBC Ab titer
Immunization
Anti-Brucella Ab titer
Dose
Increase in footpad thickness (cm -2)
-2ME
+2Me
-2Me
+2Me
0.0 0.0 0.1 0.25 0.5 1.0
N.D. a 4.8±0.4 5.3±0.6 5.5±0.0 5.2±0.3 5.8 ± 1.3
N.D. 3.7 ± 1.0 3.8±0.8 5.0±0.0 4.3±0.8 4.0±2.2
N.D. 4.0±0.7 4.7±0.6 4.7±0.6 4.0± 1.0 5.5 ±0.9
N.D. N.D. N.D. N.D. N.D. 1.0± 1.0
2.2±2.1b 0.3±0.3b
0.0 0.0 0.5
N.D. 6.0±0.0 6.7±0.6
N.D. 3.8±0.3 5.2±0.6
N.D. 3.8±0.6 4.3±0.3
N.D. N.D. N.D.
0.5±0.5 10.3 ±OA 4.0± LOb
Exp. 1 + + + + + Exp. 2 + +
1.3±OA 8.8± 1.0 8.5 ± 1.8 5.8±3.7
CDF 1 mice were iv immunized with 1 x 10 8 SRBC and 1 x 10 9 heat killed Brucella abortus cells on day O. On day 6, 1 x 109 heat killed Brucella abortus cells were secondarily iv injected. CDF 1 mice were also injected sc with 200 pI of 10mM TNBS on day 3. On day 11, 40 pI of 10mM TNBS was injected into the subcutaneous tissue of the footpads. Antibody titer was defined as agglutinating ability on day 12 and DTH was evaluated by increase in footpad thickness 24 hours after the DTH elicitation. Sera were pre-treated with 2-ME or PBS alone for 60 min at 37°C. L-156,602 was injected ip on 0,2,4,6,8 and 10 (Exp. 1) or 1,3,8 (Exp. 2). Data were shown as mean±SD. a N.D., not detected. b Statistically significant compared with immunized control mice (two tailed Studient's t-test p
