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Mechanisms of Lentinan action to peripheral blood lymphocytes from healthy persons and cancer patients
169 i n ARFC (p~O.05) and a clear improvement f o r the delayed h y p e r s e n s i t i v i t y s k i n tests (M~r i e u x score : pz.O.07). In 1 p a t i e n t who took one month's dose at one time, a cutaneous rash was noted. In 20 other patients followed f o r 6 months, we did not observe any major sideeffects.
E-N-TRIMETHYL LYSINE: A POSSIBLE ASPECIFIC IMMUNESTIMULANT B. ~lende. K. Laois. G. Elek. Zs. Suba and L. Kouoer I.Institute of Patholo~7 and Experimental Cancer Research, Medical University, Budapest, 1085, Hungary.
37 Semmelweis
I t h a s b e e n p r e v i o u s l y e v i d e n c e d by u s t h a t E - N - t r i m e t h y l l y s i n e /TML/, an amino acid derivative occurring in plants, animals and human organism, has an enhancing effect on cell proliferation. This effect is neither organ nor species specific. 200 Lug/ml o f TML causes the blastlc transformation of human peripheral b l o o ~ lymphooytes cultured in vitro. In vivo, a repeated pre- or posttreatment of mice / 5 x l O 0 m g / k g / sensitised with sheep red blood cells / s r b c / increases the hemagglutlnine titre when compared with the animals treated o n l y with srbc. On the other hand, the same TML treatment decreases the amount of food pad s w e l l i n g of sensitised mice. The intraperitoneal injection of i 0 0 m g / k g T~L increases the number of peritoneal macrophages considerably, a n d s l i g h t l y decreases the adherency of these c e l l s . T M L , as a natural and non-toxic compound, /LD50 over 2 0 0 0 m g / k g / may p o s s i b l y be used as a n i m m u n e modulant. THE EFFECTS OF F 1686, 4-PHENYL-2-(2',2',2'-TRICHLORO-ETHOXYCARBOXAMIDO)-THIAZOLE, in VARIOUS IMMUNOLOGICAL INFLAMMATORY MODELS J.P. Tarayre and H. Lauressertues Department of Pharmacology, P. Fabre Laboratory, ]7, avenue J. Moulin, 81100 CASTRES, FRANCE
38
F 1686, a new compound, increases the number of spleen cells producing hemolytic plaques towards sheep red cells, and also raises the T lymphocyte response to some mltogens such as Concanavalin A and phytohaemagglutinin. We described here the inhibition by the product of some experimental delayed hypersensitivity inflammations. The doses used generally ranged from 5 to 50 mg/kg b.w. and were far from the toxic doses in animals (LDSO p.o. in male rats : 550 mg/kg b.w. ; LD50 p.o. in male mice : 14OO mg/kg b.w.). F 1686 reduced skin hypersensitivity reactions to picryl chloride and oxazolone in mice. The intensity of action varied with the treatment modes. When given round the challenge period, the compound neatly lowered the pleural exudate and the leucocyte number at the inflammation focus in PPD (Purified Protein Derivative) hypersensitivity pleurisy in guinea-pigs. In Bordetella pertussis hypersensitivity pleurisy in rats, F 1686 steadily reduced the pleural exudate, while the action on leucocytes varied with the treatment modes. Moreover, the product had no or little effect on Freund's adjuvant polyarthritis, and on passive reversed Arthus-pleurisy and -oedema in rats. Carrageenin-induced paw oedema in rats was brought down only by high doses of the compound. The action spectrum described shows analogies with "antirheumatic" drugs (levamisole, D-penicillamine, gold salts and chloroquine).
IMMUNO-POTENTIATINGACTIVITIES OF SA-96 (2-MERCAPTO-2-METHYLPROPANOYL-L-CYSTEINE) IN VITRO I. Yamamoto and H. Ohmori Department of Medicinal Biochemistry, Faculty o f Pharmaceutical Sciences, Okayama Univers i t y , Okayama 700, Japan. 2-Mercaptoethanol (2-ME) has been widely used as an a d d i t i v e for supporting various immune responses in lymphocyte c u l t u r e s . I t has been demonstrated that 2-ME enhances primary antibody response to SRBC in v i t r o , acts as a growth-promotor of lymphoid c e l l l i n e s and stimulates the induction of c y t o t o x i c T - c e l l s . I t seems a t t r a c t i v e to note these immunop o t e n t i a t i n g a c t i v i t i e s of 2-ME in view of the p o s s i b i l i t i e s that immune responses can be regulated pharmacologically by u t i l i z i n g t h i o l compounds. SA-96 (Santen Pharmaceutical Co., L t d . , Osaka, Japan) is a novel compound synthesized r e c e n t l y which has been reported to have
39
170
suppressive effects on adjuvant a r t h r i t i s in r a t s , and c l i n i c a l a p p l i c a t i o n s are on t r i a l e s p e c i a l l y against rheumatoid a r t h r i t i s . In the present r epor t , we investigated the effects of SA-96 on various immune responses in v i t r o in comparison with those of 2-ME. SA-96, at the optimal concentration of 10-3 M, induce--6e~--mitogenicresponse in BALB/c splenic lymphocytes in the c u l t u r e containing f e t a l c a l f serum (FCS), but not in FCS-free conditions. Microscopic observations also suggested that b la s t transformation did occur in this case. On the other hand, 2-ME showed a s i g n i f i c a n t mitogenic a c t i v i t y even in FCS-free culture although much higher responses were observed in FCS-supplemented medium. SA-96 stimulated the p r o l i f e r a t i v e response to LPS s y n e r g i s t i c a l l y . I t , however, enhanced the response to Con A only a d d i t i v e l y when the lower concentration of Con A (0.25 ~g/ml) was employed, but had no e f f e c t or rather i n h i b i t o r y in the case o f higher concentrations of the mitogen. These effects were e n t i r e l y dependent on the presence of FCS in c ult ur e medium. In contrast, 2-ME enhanced p r o l i f e r a t i v e response to Con A or LPS both in the presence or absence of FCS. 2-ME (10-5 M) d r a s t i c a l l y enhanced anti-SRBC PFC response in v i t r o , while SA-96 had no e f f e c t at the concentrations of 10-5 ~I0-3 M. Both compounds, however, could stimulate polyclonal a c t i v a t i o n of mouse splenocytes by LPS which was assayed as the number of anti-SRBC PFC. These results suggest that immuno-potentiating a c t i v i t y of SA-96 would be p r e f e r e n t i a l l y directed to B - c e l l s . More d e t a i l e d i n v e s t i g a t i o n s are in progress as for the d i f f e r e n t i a l effects o f SA-96 and 2-ME on each subset of immunocompetent c e l l s .
POLYSACCHARIDES
40
AND RELATED COMPOUNDS
: LENTINAN
MECHANISMS OF LENTINAN ACTION TO PERIPHERAL BLOOD LYMPHOCYTESFROM HEALTHY PERSONSAND CANCER PATIENTS. T.Aoki, H.Miyakoshi, Y.Usuda, T.Shimizu, O.Sekine & N.Aoki. Shinrakuen Hospital, N i i g a t a s h i , JAPAN 950-21. A nonspecific immunotherapeutic agent "Lentinan", a glucan carrying molecular veight 500,000 daltons from water extracts of Lentinus edodes, was investigated to c l a r i f y i t s acting mechanisms on human peripheral blood lymphocytes (PBL) both in v i t r o and in vivo. The phytohemagglutinin (PHA)-, staphage lysate (SPL)- and concanavalin A TC-on A)-induced and spontaneous 3H-TdR incorporations of PBL from both healthy persons and cancer patients were examined by the micromethod a f t e r the in v i t r o and/or in vivo treatments with Lentinan. The IgG and IgM syntheses of human PBL were also measured in v i t r o by the solid-phase r a d i o immunoassay at varying doses of Lentinan. Since Lentinan stimulated PBL from healthy persons a t various doses in v i t r o , Lentinan may be considered as an immunopotentiator. This l y m p h o p r o l i f e r a t i o n of PBL by Lentinan was detected only when B c e l l s , T c e l l s and adherent c e l l s (mainly monocytes) coexisted. By contrast, any PBL subpopulation alone or any combination of two PBL subpopulations did not react to Lentinan. This s ti m u l a t i o n e f f e c t of Lentinan also did not detect when small numbers of PBL were cultured. Thus, both the coexistence of a l l of PBL subpopulations and the high density of PBL may be necessary f o r Lentinan s t i m u l a t i o n . Lentinan stimulated PBL from cancer patients as well as healthy persons in v i t r o before Lentinan treatment. Af t er a d m i n i s t r a t i o n of Lentinan to cancer patients, the PBL f a i l e d to react to Lentinan in v i t r o , since PBL were f u l l y activated in v i v o . Although the anti-tumor drugs had been used, the same tre:,d w~s observed. These stimulations were c l e a r l y recognized when SPL-induced and spontaneous OH-TdR incorporations were examined. When various amounts of Lentinan were added to the optimum doses of PHA, SPL and Con A f o r the examination of l y m p h o p r o l i f e r a t i v e response of PBL from healthy persons, no costimulation ef f ec t s were observed. At the lower doses of these mitogens, however, varying amounts of Lentinan added did s l i g h t l y change the l y m p h o p r o l i f e r a t i v e response, g e n e r a l l y showing an increase, except f o r a l i t t l e decrease in the case of PHA. The SPL- and PWM-induced IgG and IgM syntheses of PBL from healthy persons were augmented in v i t r o ~t various doses of Lentinan. These changes were not related to those of s p o n t a ~ JH-TdR incorporation of PBL.
41
Lentinan As A Distinct Immunopotentiator J. Hamuro, M. Ta~arada, N. Kashima¢ Y. Akiyama r K. Izawa, K. Mitsugi Central Research Laboratories, Ajinomoto Co., Inc., Yokohama Japan A distinct T cell specific immune adjuvant, Lentinan, posessed several relevant characteristics as compared with other high mol.wt, immune adjuvants, such as zymosan,
E-N-TRIMETHYL LYSINE: A POSSIBLE ASPECIFIC IMMUNESTIMULANT B. ~lende. K. Laois. G. Elek. Zs. Suba and L. Kouoer I.Institute of Patholo~7 and Experimental Cancer Research, Medical University, Budapest, 1085, Hungary.
37 Semmelweis
I t h a s b e e n p r e v i o u s l y e v i d e n c e d by u s t h a t E - N - t r i m e t h y l l y s i n e /TML/, an amino acid derivative occurring in plants, animals and human organism, has an enhancing effect on cell proliferation. This effect is neither organ nor species specific. 200 Lug/ml o f TML causes the blastlc transformation of human peripheral b l o o ~ lymphooytes cultured in vitro. In vivo, a repeated pre- or posttreatment of mice / 5 x l O 0 m g / k g / sensitised with sheep red blood cells / s r b c / increases the hemagglutlnine titre when compared with the animals treated o n l y with srbc. On the other hand, the same TML treatment decreases the amount of food pad s w e l l i n g of sensitised mice. The intraperitoneal injection of i 0 0 m g / k g T~L increases the number of peritoneal macrophages considerably, a n d s l i g h t l y decreases the adherency of these c e l l s . T M L , as a natural and non-toxic compound, /LD50 over 2 0 0 0 m g / k g / may p o s s i b l y be used as a n i m m u n e modulant. THE EFFECTS OF F 1686, 4-PHENYL-2-(2',2',2'-TRICHLORO-ETHOXYCARBOXAMIDO)-THIAZOLE, in VARIOUS IMMUNOLOGICAL INFLAMMATORY MODELS J.P. Tarayre and H. Lauressertues Department of Pharmacology, P. Fabre Laboratory, ]7, avenue J. Moulin, 81100 CASTRES, FRANCE
38
F 1686, a new compound, increases the number of spleen cells producing hemolytic plaques towards sheep red cells, and also raises the T lymphocyte response to some mltogens such as Concanavalin A and phytohaemagglutinin. We described here the inhibition by the product of some experimental delayed hypersensitivity inflammations. The doses used generally ranged from 5 to 50 mg/kg b.w. and were far from the toxic doses in animals (LDSO p.o. in male rats : 550 mg/kg b.w. ; LD50 p.o. in male mice : 14OO mg/kg b.w.). F 1686 reduced skin hypersensitivity reactions to picryl chloride and oxazolone in mice. The intensity of action varied with the treatment modes. When given round the challenge period, the compound neatly lowered the pleural exudate and the leucocyte number at the inflammation focus in PPD (Purified Protein Derivative) hypersensitivity pleurisy in guinea-pigs. In Bordetella pertussis hypersensitivity pleurisy in rats, F 1686 steadily reduced the pleural exudate, while the action on leucocytes varied with the treatment modes. Moreover, the product had no or little effect on Freund's adjuvant polyarthritis, and on passive reversed Arthus-pleurisy and -oedema in rats. Carrageenin-induced paw oedema in rats was brought down only by high doses of the compound. The action spectrum described shows analogies with "antirheumatic" drugs (levamisole, D-penicillamine, gold salts and chloroquine).
IMMUNO-POTENTIATINGACTIVITIES OF SA-96 (2-MERCAPTO-2-METHYLPROPANOYL-L-CYSTEINE) IN VITRO I. Yamamoto and H. Ohmori Department of Medicinal Biochemistry, Faculty o f Pharmaceutical Sciences, Okayama Univers i t y , Okayama 700, Japan. 2-Mercaptoethanol (2-ME) has been widely used as an a d d i t i v e for supporting various immune responses in lymphocyte c u l t u r e s . I t has been demonstrated that 2-ME enhances primary antibody response to SRBC in v i t r o , acts as a growth-promotor of lymphoid c e l l l i n e s and stimulates the induction of c y t o t o x i c T - c e l l s . I t seems a t t r a c t i v e to note these immunop o t e n t i a t i n g a c t i v i t i e s of 2-ME in view of the p o s s i b i l i t i e s that immune responses can be regulated pharmacologically by u t i l i z i n g t h i o l compounds. SA-96 (Santen Pharmaceutical Co., L t d . , Osaka, Japan) is a novel compound synthesized r e c e n t l y which has been reported to have
39
170
suppressive effects on adjuvant a r t h r i t i s in r a t s , and c l i n i c a l a p p l i c a t i o n s are on t r i a l e s p e c i a l l y against rheumatoid a r t h r i t i s . In the present r epor t , we investigated the effects of SA-96 on various immune responses in v i t r o in comparison with those of 2-ME. SA-96, at the optimal concentration of 10-3 M, induce--6e~--mitogenicresponse in BALB/c splenic lymphocytes in the c u l t u r e containing f e t a l c a l f serum (FCS), but not in FCS-free conditions. Microscopic observations also suggested that b la s t transformation did occur in this case. On the other hand, 2-ME showed a s i g n i f i c a n t mitogenic a c t i v i t y even in FCS-free culture although much higher responses were observed in FCS-supplemented medium. SA-96 stimulated the p r o l i f e r a t i v e response to LPS s y n e r g i s t i c a l l y . I t , however, enhanced the response to Con A only a d d i t i v e l y when the lower concentration of Con A (0.25 ~g/ml) was employed, but had no e f f e c t or rather i n h i b i t o r y in the case o f higher concentrations of the mitogen. These effects were e n t i r e l y dependent on the presence of FCS in c ult ur e medium. In contrast, 2-ME enhanced p r o l i f e r a t i v e response to Con A or LPS both in the presence or absence of FCS. 2-ME (10-5 M) d r a s t i c a l l y enhanced anti-SRBC PFC response in v i t r o , while SA-96 had no e f f e c t at the concentrations of 10-5 ~I0-3 M. Both compounds, however, could stimulate polyclonal a c t i v a t i o n of mouse splenocytes by LPS which was assayed as the number of anti-SRBC PFC. These results suggest that immuno-potentiating a c t i v i t y of SA-96 would be p r e f e r e n t i a l l y directed to B - c e l l s . More d e t a i l e d i n v e s t i g a t i o n s are in progress as for the d i f f e r e n t i a l effects o f SA-96 and 2-ME on each subset of immunocompetent c e l l s .
POLYSACCHARIDES
40
AND RELATED COMPOUNDS
: LENTINAN
MECHANISMS OF LENTINAN ACTION TO PERIPHERAL BLOOD LYMPHOCYTESFROM HEALTHY PERSONSAND CANCER PATIENTS. T.Aoki, H.Miyakoshi, Y.Usuda, T.Shimizu, O.Sekine & N.Aoki. Shinrakuen Hospital, N i i g a t a s h i , JAPAN 950-21. A nonspecific immunotherapeutic agent "Lentinan", a glucan carrying molecular veight 500,000 daltons from water extracts of Lentinus edodes, was investigated to c l a r i f y i t s acting mechanisms on human peripheral blood lymphocytes (PBL) both in v i t r o and in vivo. The phytohemagglutinin (PHA)-, staphage lysate (SPL)- and concanavalin A TC-on A)-induced and spontaneous 3H-TdR incorporations of PBL from both healthy persons and cancer patients were examined by the micromethod a f t e r the in v i t r o and/or in vivo treatments with Lentinan. The IgG and IgM syntheses of human PBL were also measured in v i t r o by the solid-phase r a d i o immunoassay at varying doses of Lentinan. Since Lentinan stimulated PBL from healthy persons a t various doses in v i t r o , Lentinan may be considered as an immunopotentiator. This l y m p h o p r o l i f e r a t i o n of PBL by Lentinan was detected only when B c e l l s , T c e l l s and adherent c e l l s (mainly monocytes) coexisted. By contrast, any PBL subpopulation alone or any combination of two PBL subpopulations did not react to Lentinan. This s ti m u l a t i o n e f f e c t of Lentinan also did not detect when small numbers of PBL were cultured. Thus, both the coexistence of a l l of PBL subpopulations and the high density of PBL may be necessary f o r Lentinan s t i m u l a t i o n . Lentinan stimulated PBL from cancer patients as well as healthy persons in v i t r o before Lentinan treatment. Af t er a d m i n i s t r a t i o n of Lentinan to cancer patients, the PBL f a i l e d to react to Lentinan in v i t r o , since PBL were f u l l y activated in v i v o . Although the anti-tumor drugs had been used, the same tre:,d w~s observed. These stimulations were c l e a r l y recognized when SPL-induced and spontaneous OH-TdR incorporations were examined. When various amounts of Lentinan were added to the optimum doses of PHA, SPL and Con A f o r the examination of l y m p h o p r o l i f e r a t i v e response of PBL from healthy persons, no costimulation ef f ec t s were observed. At the lower doses of these mitogens, however, varying amounts of Lentinan added did s l i g h t l y change the l y m p h o p r o l i f e r a t i v e response, g e n e r a l l y showing an increase, except f o r a l i t t l e decrease in the case of PHA. The SPL- and PWM-induced IgG and IgM syntheses of PBL from healthy persons were augmented in v i t r o ~t various doses of Lentinan. These changes were not related to those of s p o n t a ~ JH-TdR incorporation of PBL.
41
Lentinan As A Distinct Immunopotentiator J. Hamuro, M. Ta~arada, N. Kashima¢ Y. Akiyama r K. Izawa, K. Mitsugi Central Research Laboratories, Ajinomoto Co., Inc., Yokohama Japan A distinct T cell specific immune adjuvant, Lentinan, posessed several relevant characteristics as compared with other high mol.wt, immune adjuvants, such as zymosan,