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Interferon-induced macrophages cytolytic activity against tumor cells
157
After
in
significant ficant
vitro increase
effect The
stimulating
incubation
was
results
of
E-R
observed were
similar
with and in
of
all
drugs
we
observed
LAI
test
while
no
polymorphonuc!ear when
employing
a
signi-
phagocy~osis. different
immuno-
agents.
SERUM THYMIC FACTOR DETERMINATION
lOa
IN DIFFERENT HUMAN PATHOLOGIES
N. Fabris In~nunol Ctr INRCA Res. Dept. - 60100 Ancona - Italy Serum thymic factors (FTS) has been measured with the meted of Bach and coll. in patients suffering from pathological conditions frequently accompanied by manifestations of peripheral reduced ir~nunological capacity. These human pathologies included: a) diseases with a genetic background, such as trisomy -21, congenital muscular athrophy (Duchenne syndrome) and hereditary susceptibility to allergic phenomena; b) heterogenous diseases, mainly due to traumatic or surgical cerebral lesions, with the conmlon denominator to oblige patients to undergo reanimation intensive therapy. Findings on the first group of diseases revealed that in the majority of Dowr~ (12 out 16 subjects) and of athopic individuals (iO out 15 subjects) and in all Duchen~e pa~tients (5 subjects) the circulating level of FTS was significsntly lower than that recorded in age and sex-matched normal individuals. In Down syndrome, but not in athopic individuals, the percentual of subjects with low level of FTS is higher in the group of i-iO years of age than in older groups. The level of FTS in patients under reanimation intensive therapy is quite variable within the group: 5 ~ of these patients (9 out of 17) show, however, a level of FTS much lower than that expected by their age, Wh~le these findings Glearly demonstrate that the rate of synthesis or relase of FTS may depend on a number of regulatory factors, which, presumably,are disturbed in some human pathologies, further investigations are needed in order to know the causes of such FTS variations and the relevance that they have for the peripheral efficiency of the irmmune system. In all cases, due to the observed variabily of FTS level within a single syndrome it seems reasonable to propose the determination of FTS level as abligatory propedeutical to any trial of substitutive therapy with thymic hormone preparation.
INTERFERON A N D INDUCERS INTERFERON-INDUCED MACROPHAGES CYTOLYTIC ACTIVITY AGAINST TUMOR CELLS D. Boraschi, S. Alberti, F. Spreafico and A. Tagliabue Istituto di Ricerche Farmacologiche "Marie Negri" - Via Eritrea, 62 - 20157 MILAN,
11 Italy
Mouse peritoneal macrophages can be activated to express cytotoxic activity (either cytostasis or cytolysis) against tumor cells by a variety of in vivo (BCG, C.parvum, pyran) or in vitro (lymphokines, LPS) stimuli. Macrophages exposed to partially purified fibroblast interferon (IF) develop the ability of lysing tumor cells, as measured in a 48-72 h in vitro 3H-thymidine release assay. IF-treated macrophages also express cytostatic activity against tumor cells. IF-induced macrophage cytotoxicity is optimal when doses of IOO-IOO0 U/ml of IF and exposure times of 6 to 24 h are used for in vitro activation. No activation of mouse peritoneal macrophages can be achieved by using human IF (which enhances tumoricidal capability of human monocytes) or mouse mock IF as in vitro stimuli. The mechanism of macrophage activation by IF has also been examined, in order to ascertain whether activation by IF follows the same pathway of that by MAF (macrophage activation factor). It is in fact described that MAF triggers macrophage cytotoxicity by a two-step mechanism, first providing a preparation signal to the cells and then a second, expression signal (Ruco and Meltzer, J.Immunol. 121: 2035, 1978). IF and MAF have been therefore compared in their ability to
158
provide such signals to macrophages. First signal activity has been studied in two mouse strains: the C3H/HeN mice, routinely used in all the experiments, and the genetically related C3H/HeJ mice, the defective model in which the two-step activation mechanism had been studied. Macrophages from either strain were treated with IF in presence of LPS (as second signal). Whereas C3H/HeN macrophages treated with IF and LPS develop strong cytotoxic activity, C3H/HeJ cells are not activated. MAF with LPS, on the other hand, induces strong cytotoxicity also in C3H/HeJ macrophages. The IF ability of acting as second signal has been examined by priming macrophages with suboptimal doses of MAF (which acts as first signal) and then adding IF. IF does not trigger the primed macrophages to express cytotoxic activity, i.e. it does not act as second activation signal. This pattern of activity thus suggests for IF a different activation mechanism, which does not include the two signals of MAF activation. IF-induced macrophage cytotoxicity therefore represents a very interesting model for the analysis of mechanisms of macrophage activation.
12
MITOGENIC SUBSTANCES IN STAPHAGE LYSATE (SPL). H.Miyakoshi*, T.Aoki* & M.Mizukoshi** *Research D i v i s i o n , Shinrakuen Hospital, Nishiariakecho 1-27, N i i g a t a s h i , JAPAN 950-21. **Immunology D i v i s i o n , Research Center, Fujizoki Pharmatheutical Co.,Ltd.,Tokyo, JAPAN. Since the i n i t i n a l production of staphylococcus phage lysate (staphage lysate = SPL), consisting of components of Staphylococcus aureus, Serotypes I and m, lysed by a polyvalent staphylococcus bacteriophage, SPL has been u t i l i z e d in the c l i n i c to t r e a t a v a r i e t y of diseases, e . g . , i n f e c t i o u s diseases including v i r a l and bacterial i n f e c t i o n s , Crohn's disease, bronchial asthma, and malignant diseases. As a mitogen, SPL can markedly stimulate peripheral blood lymphocytes (PBL) from humanbeings. The subpopulations of PBL stimulated by SPL are T c e l l s , B c e l l s and null c e l l s . Although SPL is known to contain staphylococcus components and active bacteriophage, i t remains unknown which of these substances is mitogenic. We have t r i e d to answer to t h i s question mainly using 3H-TdR incorporation of PBL from healthy persons by the micromethod a f t e r 5 days incubation. The SPL-induced IgG and IgM syntheses-of PBL were also measured by the solid-phase radioimmunoassay. A f t e r removal of bacteriophage by u l t r a c e n t r i f u g a t i o n (twice at 200,000 xg f o r 3 hrs), the supernatant revealed the l y m p h o p r o l i f e r a t i v e response of PBL. On the other hand, the viable bacteriophage f a i l e d to stimulate PBL. In a d d i t i o n , the protein A f r a c t i o n did not c o n t r i b u t e to the mitogenic a c t i v i t y of SPL, although i t has been reported that the protein A from S. aureus exhibited m i t o g e n i c i t y in human PBL. The molecular weight of the mitogenic substance was approximately 25,000 daltons due to the f r a c t i o n a t i o n with Sephadex G-50 Superfine and Amicon F i l t e r s . S.aureus secreted the mitogenic substance into the c u l t u r e medium without adding the bacteriophage. Destroyed S. aureus by sonication at 9,000 cycles /sec f o r 1 min also possessed the mitogenic a c t i v i t y . By contrast, the c u l t u r e medium (beef heart i n f u s i o n broth) i t s e l f displayed no l y m p h o p r o l i f e r a t i v e a c t i v i t y . SPL was able to stimulate the IgG and IgM syntheses 4- to 20-fold in comparison with the c o n t r o l . The protein A-free f r a c t i o n and bacteriophage-free f r a c t i o n were also able to induce the IgG and IgM syntheses of PBL.
13
HEPATIC MICROSOMAL ENZYME ACTIVITY AND EXPRESSION TREATED WITH THE IMMUNOSTIMULANT PYRAN COPOLYMER P. Puccetti, Giampietri A. and Contessa A. R. Institute of Pharmacology, University of Perugia,
OF NATURAL CYTOTOXICITY
08100 Perugia,
IN MICE
Italy.
The spontaneous in vitro expression of natural killer iNK] cell activity against tumor targets is known to be affected by a variety of both endogenous and exogenous factors and the biological half-life of these substances may be relevant to the levels of NK expression. Natural cytotoxicity is greatly reduced in mice at 5-7 days of administration of the synthet ic polyanion pyran copolymer [Puccetti et al., Int. J. Cancer: 24, 81B, 197B). This agent was shown in preliminary experiments to remarkably diminish the activity of murine hepatic microsomal enzymes (HME] , an action possiblycausil~g major changes in serum levels of many endogenous metabolites. In the present study the possibility was explored of a correlation between HME activity and the levels of NK expression in the mouse. Liver microsome activity was assessed in vitro in terms of aminopyrine N-demethylase and aniline hydroxylation activities whereas NK-cell mediated cytolysis was measured in a short-term 51Cr release assay against YAC-1 target cells. A single injection of pyran copolymer (25-225 mg/kg, ip) caused a dose-dependent inhibition of HME activity, which lasted at least 10 days and reached peak levels at 5-7 days, at a time when suppression of NK cell activity was max-
After
in
significant ficant
vitro increase
effect The
stimulating
incubation
was
results
of
E-R
observed were
similar
with and in
of
all
drugs
we
observed
LAI
test
while
no
polymorphonuc!ear when
employing
a
signi-
phagocy~osis. different
immuno-
agents.
SERUM THYMIC FACTOR DETERMINATION
lOa
IN DIFFERENT HUMAN PATHOLOGIES
N. Fabris In~nunol Ctr INRCA Res. Dept. - 60100 Ancona - Italy Serum thymic factors (FTS) has been measured with the meted of Bach and coll. in patients suffering from pathological conditions frequently accompanied by manifestations of peripheral reduced ir~nunological capacity. These human pathologies included: a) diseases with a genetic background, such as trisomy -21, congenital muscular athrophy (Duchenne syndrome) and hereditary susceptibility to allergic phenomena; b) heterogenous diseases, mainly due to traumatic or surgical cerebral lesions, with the conmlon denominator to oblige patients to undergo reanimation intensive therapy. Findings on the first group of diseases revealed that in the majority of Dowr~ (12 out 16 subjects) and of athopic individuals (iO out 15 subjects) and in all Duchen~e pa~tients (5 subjects) the circulating level of FTS was significsntly lower than that recorded in age and sex-matched normal individuals. In Down syndrome, but not in athopic individuals, the percentual of subjects with low level of FTS is higher in the group of i-iO years of age than in older groups. The level of FTS in patients under reanimation intensive therapy is quite variable within the group: 5 ~ of these patients (9 out of 17) show, however, a level of FTS much lower than that expected by their age, Wh~le these findings Glearly demonstrate that the rate of synthesis or relase of FTS may depend on a number of regulatory factors, which, presumably,are disturbed in some human pathologies, further investigations are needed in order to know the causes of such FTS variations and the relevance that they have for the peripheral efficiency of the irmmune system. In all cases, due to the observed variabily of FTS level within a single syndrome it seems reasonable to propose the determination of FTS level as abligatory propedeutical to any trial of substitutive therapy with thymic hormone preparation.
INTERFERON A N D INDUCERS INTERFERON-INDUCED MACROPHAGES CYTOLYTIC ACTIVITY AGAINST TUMOR CELLS D. Boraschi, S. Alberti, F. Spreafico and A. Tagliabue Istituto di Ricerche Farmacologiche "Marie Negri" - Via Eritrea, 62 - 20157 MILAN,
11 Italy
Mouse peritoneal macrophages can be activated to express cytotoxic activity (either cytostasis or cytolysis) against tumor cells by a variety of in vivo (BCG, C.parvum, pyran) or in vitro (lymphokines, LPS) stimuli. Macrophages exposed to partially purified fibroblast interferon (IF) develop the ability of lysing tumor cells, as measured in a 48-72 h in vitro 3H-thymidine release assay. IF-treated macrophages also express cytostatic activity against tumor cells. IF-induced macrophage cytotoxicity is optimal when doses of IOO-IOO0 U/ml of IF and exposure times of 6 to 24 h are used for in vitro activation. No activation of mouse peritoneal macrophages can be achieved by using human IF (which enhances tumoricidal capability of human monocytes) or mouse mock IF as in vitro stimuli. The mechanism of macrophage activation by IF has also been examined, in order to ascertain whether activation by IF follows the same pathway of that by MAF (macrophage activation factor). It is in fact described that MAF triggers macrophage cytotoxicity by a two-step mechanism, first providing a preparation signal to the cells and then a second, expression signal (Ruco and Meltzer, J.Immunol. 121: 2035, 1978). IF and MAF have been therefore compared in their ability to
158
provide such signals to macrophages. First signal activity has been studied in two mouse strains: the C3H/HeN mice, routinely used in all the experiments, and the genetically related C3H/HeJ mice, the defective model in which the two-step activation mechanism had been studied. Macrophages from either strain were treated with IF in presence of LPS (as second signal). Whereas C3H/HeN macrophages treated with IF and LPS develop strong cytotoxic activity, C3H/HeJ cells are not activated. MAF with LPS, on the other hand, induces strong cytotoxicity also in C3H/HeJ macrophages. The IF ability of acting as second signal has been examined by priming macrophages with suboptimal doses of MAF (which acts as first signal) and then adding IF. IF does not trigger the primed macrophages to express cytotoxic activity, i.e. it does not act as second activation signal. This pattern of activity thus suggests for IF a different activation mechanism, which does not include the two signals of MAF activation. IF-induced macrophage cytotoxicity therefore represents a very interesting model for the analysis of mechanisms of macrophage activation.
12
MITOGENIC SUBSTANCES IN STAPHAGE LYSATE (SPL). H.Miyakoshi*, T.Aoki* & M.Mizukoshi** *Research D i v i s i o n , Shinrakuen Hospital, Nishiariakecho 1-27, N i i g a t a s h i , JAPAN 950-21. **Immunology D i v i s i o n , Research Center, Fujizoki Pharmatheutical Co.,Ltd.,Tokyo, JAPAN. Since the i n i t i n a l production of staphylococcus phage lysate (staphage lysate = SPL), consisting of components of Staphylococcus aureus, Serotypes I and m, lysed by a polyvalent staphylococcus bacteriophage, SPL has been u t i l i z e d in the c l i n i c to t r e a t a v a r i e t y of diseases, e . g . , i n f e c t i o u s diseases including v i r a l and bacterial i n f e c t i o n s , Crohn's disease, bronchial asthma, and malignant diseases. As a mitogen, SPL can markedly stimulate peripheral blood lymphocytes (PBL) from humanbeings. The subpopulations of PBL stimulated by SPL are T c e l l s , B c e l l s and null c e l l s . Although SPL is known to contain staphylococcus components and active bacteriophage, i t remains unknown which of these substances is mitogenic. We have t r i e d to answer to t h i s question mainly using 3H-TdR incorporation of PBL from healthy persons by the micromethod a f t e r 5 days incubation. The SPL-induced IgG and IgM syntheses-of PBL were also measured by the solid-phase radioimmunoassay. A f t e r removal of bacteriophage by u l t r a c e n t r i f u g a t i o n (twice at 200,000 xg f o r 3 hrs), the supernatant revealed the l y m p h o p r o l i f e r a t i v e response of PBL. On the other hand, the viable bacteriophage f a i l e d to stimulate PBL. In a d d i t i o n , the protein A f r a c t i o n did not c o n t r i b u t e to the mitogenic a c t i v i t y of SPL, although i t has been reported that the protein A from S. aureus exhibited m i t o g e n i c i t y in human PBL. The molecular weight of the mitogenic substance was approximately 25,000 daltons due to the f r a c t i o n a t i o n with Sephadex G-50 Superfine and Amicon F i l t e r s . S.aureus secreted the mitogenic substance into the c u l t u r e medium without adding the bacteriophage. Destroyed S. aureus by sonication at 9,000 cycles /sec f o r 1 min also possessed the mitogenic a c t i v i t y . By contrast, the c u l t u r e medium (beef heart i n f u s i o n broth) i t s e l f displayed no l y m p h o p r o l i f e r a t i v e a c t i v i t y . SPL was able to stimulate the IgG and IgM syntheses 4- to 20-fold in comparison with the c o n t r o l . The protein A-free f r a c t i o n and bacteriophage-free f r a c t i o n were also able to induce the IgG and IgM syntheses of PBL.
13
HEPATIC MICROSOMAL ENZYME ACTIVITY AND EXPRESSION TREATED WITH THE IMMUNOSTIMULANT PYRAN COPOLYMER P. Puccetti, Giampietri A. and Contessa A. R. Institute of Pharmacology, University of Perugia,
OF NATURAL CYTOTOXICITY
08100 Perugia,
IN MICE
Italy.
The spontaneous in vitro expression of natural killer iNK] cell activity against tumor targets is known to be affected by a variety of both endogenous and exogenous factors and the biological half-life of these substances may be relevant to the levels of NK expression. Natural cytotoxicity is greatly reduced in mice at 5-7 days of administration of the synthet ic polyanion pyran copolymer [Puccetti et al., Int. J. Cancer: 24, 81B, 197B). This agent was shown in preliminary experiments to remarkably diminish the activity of murine hepatic microsomal enzymes (HME] , an action possiblycausil~g major changes in serum levels of many endogenous metabolites. In the present study the possibility was explored of a correlation between HME activity and the levels of NK expression in the mouse. Liver microsome activity was assessed in vitro in terms of aminopyrine N-demethylase and aniline hydroxylation activities whereas NK-cell mediated cytolysis was measured in a short-term 51Cr release assay against YAC-1 target cells. A single injection of pyran copolymer (25-225 mg/kg, ip) caused a dose-dependent inhibition of HME activity, which lasted at least 10 days and reached peak levels at 5-7 days, at a time when suppression of NK cell activity was max-