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Immunokinetics of a single dose of thymopoietin pentapeptide
154
3
STIMULATION MICE
OF T H Y M I C
HUMORAL
M.Dardenne~
P.Niaudet2,N.Simon-Lavoine
I I N S E R M U 25 - H S p i t a l 2 I N S E R M U 30 A
single
injection
(250~g/mouse
i.p.)
crease
level.
in F T S
jection
and
observed
Th earliest
90 to
level 150.
back
to n o r m a l
in a g e i n g
mice
and
is s e e n
animals.
The
appearance mice,
and
adsorbant
thymic
o f FTS
activity
in v i t r o
after
purified
attempt
animals
dependence
to e s t a b l i s h
its c o n s e q u e n c e s
mice
antibody
on
than
with or
been
after
the
of
is
FTS.
thymic
in-
is and
is o b s e r v e d
Conversely,
no v a r i a -
thymectomized
assessed
by t h e d i s -
of c y c l o m u n i n e ~ r e a t e d
serum with
demonstrated
value)
level
in-
after
progressively
in FTS
kinetics.
increase
peptide
14 d a y s
normal
decrease
thymectomy
of the
synthetic
the mechanisms
IN
France
and p r o g r e s s i v e
is n o t e d
increase
variations
antibodies.The
has
a rapid
rise
i00 t i m e s
similar
Neuilly/Seine
in C y c l o m u n i n e - i n j e c t e d
in v i v o
PEPTIDE,
immunomodulating
mice
then
same
of FTS
against
immune
young
levels The
incubation
of a n t i - F T S
cyclomunine-treated using
FTS
range.
A CYCLIC
Servier,
a cyclic
significant
(more
in N Z B m i c e
in c o n t r o l
made
3 Institut
i n d u c e s in n o r m a l
the m a x i m u m
on d a y s
,Paris
BY C Y C L O M U N I N E , 3, and J . F . B a c h 1
of C y c l o m u n i n e ,
stabilize
tion
4
Necker
FUNCTION
a FTS-specific
in thymic
by i n d i r e c t Studies
by
immunofluorescence
in p r o g r e s s
stimulation
immuno-
FTS s e c r e t i o n
will
by c y c l o m u n i n e
and
function
EFFECT OF A THYMIC FACTOR (TS') ON THE IMMUNE RESPONSE OF AGED GUINEA FIGS R. Falchetti, C. Cafiero, L. Caprino Department of Pharmacology - Istituto Farmacologico Serono,Rome - Italy The in vivo administration of a thymic extract (TS), in neonatal guinea pigs, in which the T-cell subpopulations express different levels of functional activity, was found, in a previous work, to differently influence the immunological responsiveness or maturation stake of the lymphoid spleen cells, according to the function studied. TS administration induced an increase in the number of antibody-producing cells, an increase in the response to mitogenie stinm/lation but no variation in the number of E-rosette forming cells. The present work illustrates the activity of TS on aged guinea pigs. TS was found, in i_nn vitro experiments to increase both the response of peripheral blood lymphocytes from aged animals to lectins, and the number of E-rosette forming cells. Furthermore the in vivo administration of TS to the aged animals resulted in a restoration of immunocompetence of T-cells as shown by the increase of the response of spleen lymphoid cells %0 sheep red blood cells and to specific T-cell antigens stimulation. I) TS = Thymostimulin - In previous papers the same product was named as TP-I
5
IMMUNOKINETICS OF A SINGLE DOSE OF THYMOPOIETIN PENTAPEPTIDE. F. La~hi Pasini~ A, Auteri~ A.L. Pasqui and T. Di Perri Department of Internal Medicine s School of Medicine~ University of Siena, 53100 Siena Italy. Previous studies have shown that 'Tin vitro" incubation with Thymopoietin pentapeptide(TPS) is able to normalize the number of E-rosette forming cells in Rheumatoid Arthritis (RA) patients when depressed. According to these premises 14 patients with classical or definite
155
RA and a low number of E-rosette forming cells were given single doses of 5 mg and 50 mg of TP5 intravenously in order to verify the "in vivo" effect of the drug. TP5 produced a dose-related sustained increase in E-rosette forming lymphocytes as shown by the decrease of thymopoietin dependent rosetting ratio. A single dose of 50 mg restored the n ~ b e r of E-rosette forming cells to normal after 12h and this effect lasted 6 to 7 days. With the administration of 5 mg of thymopoietin pentapeptide, the basal values of E-rosette forming ~;ells markedly increased after 12h and returned to the pretreatment level on the 2nd day. These immunokinetic data clarify some aspects of the clinical pharmacology of TP5 and must be considered for a rational schedule of treatment with the drug.
ISOLATION AND ASSAY OF SPECIFIC T-LYMPHOCYTE PROLIFERATION INHIBITORS (CHALONES) FROM CALF THYMUS H.R. Maurer and R.Maschler Pharmazeutisches Institut der Freien Universit~t Berlin K8nigin-Luise-StraBe 2+4, D-!O00 Berlin 33, Germany
6
Increasing evidence sugges~that the proliferation of activated T-lymphocytes is regulated not only by stimulating (e.g. T-cell growth factor), but also by inhibiting factors. Some of the latter have been found to suppress T-lymphocyte proliferation in a cell-line specific,non-toxic and reversible manner and may therefore be classified as chalones. Using agar colony assays in glass capillary tubes with truly proliferating, stimulating human T-lymphocytes and mouse granulocytes, we isolated from calf thymus acetone powder 2 ultrafiltrate fractions with chalone properties: F! in the MW range l-!OxlO- proved to ~e stable upon heating, prolonged storage and lyophilization, whereas F2 in the range !-3xlO was found to be unstable. Tween 80 and cetyltrimethylanm~onium bromide facilitated the extraction of F2 and F2. Biogel P6 chromatography of FI increased the specificity of inhibition of lymphocyte colony growt h and revealed an MW of approx. 1.400d.Upon DEAE cellulose chromatography of the active pool an inhibitor of high specific activity was eluted with 0.2 M NaCI, devoid of any granulopoiesis inhibitor. Using a washing procedure prior to the colony assay we demonstrated its reversibility of action as well as its non-toxicity. Its activity in the nM-range was found unlikely to result from spermine, but from an anionic peptide. Loss of activity upon storage due to adsorption, varying yields from 2 kg tissue batches and the low quantities ( n g - ~g) of the purified inhibitor require large scale isolation procedures which are underway. In summary, a T-lymphocyte chalone of high specific activity was isolated acting as an antigen-nonspecifie suppressor factor of T-cell proliferation dependent immune response.
ROLE OF I~{E THYTqUS IN THE ~{0DUCTION OF THE HO~,~O~£ DIFFERENTIATING ~qD ~TURIIIG T CELLS G. Renoux I ~i. Renoux and J.M. Guil1~umin Laboratoire d'Immunologie, Facult6 de M6decine, B.P. 5223, 37052 Tours Cedex, FRANCE.
The t h y m u s f r o m a f e m a l e C 5 7 B L / 6 ( B 6 ) m o u s e was i n t r a p e r i t o n e a l l y g r a f t e d t o a f e m a l e n_~/nu (B6 b a c k g r o u n d ) m o u s e w h i c h was t r e a t e d w i t h 25 m g / k g DTC, o r w i t h s a l i n e a s c o n t r o l , 15 d a y s later. S e r u m wa~ s a m p l e d 24 h l a t e r , h e a t e d a t 56°C f o r ~5 m i n , a n d t e s t e d f o r i t s c a p a c i t y t o i n d u c e T h y - 1 - c e l l s i n n_~/n~u s p l e e n c e l l s , a f t e r 7 . 5 h - i n c u b a t i o n , in a microcytotoxicity a s s a y u s i n g m o n o c l o n a l ~ - T h y - 1 a n t i b o d y . D a t a w e r e c o m p a r e d w i t h t h e e f f e c t o f ~11C on n o r m a l t h y m u s - b e a r i n g B6 m i c e . E a c h g r o u p c o m p r i z e d a t l e a s t 5 m i c e . A s y n g e n e i c t h y m u s g r a f t i n d u c e s i n t h e n_~/nu m o u s e t h e p r o d u c t i o n o f a p a r t o f t h e h o r m o n a l a c t i v i t y found in normal B6 mice. A single administration of DTC triggers in n_~/nu B6 the synthesis off a fully-active hormone. When the serum of thymus-reconstituted and DTC-treated nude mice was tested, the dose response our~e paralleled that of the serum from normal B6 mice treated with DTC, but at markedly lower thresholds of T-cell inducing capacity. A cellular system other than the thymus synthetizes under the influence of DTC the hormone specifically active on the T-cell lineage. The thymus appears as the organ which regulates and controls the production of this hormone.
7
3
STIMULATION MICE
OF T H Y M I C
HUMORAL
M.Dardenne~
P.Niaudet2,N.Simon-Lavoine
I I N S E R M U 25 - H S p i t a l 2 I N S E R M U 30 A
single
injection
(250~g/mouse
i.p.)
crease
level.
in F T S
jection
and
observed
Th earliest
90 to
level 150.
back
to n o r m a l
in a g e i n g
mice
and
is s e e n
animals.
The
appearance mice,
and
adsorbant
thymic
o f FTS
activity
in v i t r o
after
purified
attempt
animals
dependence
to e s t a b l i s h
its c o n s e q u e n c e s
mice
antibody
on
than
with or
been
after
the
of
is
FTS.
thymic
in-
is and
is o b s e r v e d
Conversely,
no v a r i a -
thymectomized
assessed
by t h e d i s -
of c y c l o m u n i n e ~ r e a t e d
serum with
demonstrated
value)
level
in-
after
progressively
in FTS
kinetics.
increase
peptide
14 d a y s
normal
decrease
thymectomy
of the
synthetic
the mechanisms
IN
France
and p r o g r e s s i v e
is n o t e d
increase
variations
antibodies.The
has
a rapid
rise
i00 t i m e s
similar
Neuilly/Seine
in C y c l o m u n i n e - i n j e c t e d
in v i v o
PEPTIDE,
immunomodulating
mice
then
same
of FTS
against
immune
young
levels The
incubation
of a n t i - F T S
cyclomunine-treated using
FTS
range.
A CYCLIC
Servier,
a cyclic
significant
(more
in N Z B m i c e
in c o n t r o l
made
3 Institut
i n d u c e s in n o r m a l
the m a x i m u m
on d a y s
,Paris
BY C Y C L O M U N I N E , 3, and J . F . B a c h 1
of C y c l o m u n i n e ,
stabilize
tion
4
Necker
FUNCTION
a FTS-specific
in thymic
by i n d i r e c t Studies
by
immunofluorescence
in p r o g r e s s
stimulation
immuno-
FTS s e c r e t i o n
will
by c y c l o m u n i n e
and
function
EFFECT OF A THYMIC FACTOR (TS') ON THE IMMUNE RESPONSE OF AGED GUINEA FIGS R. Falchetti, C. Cafiero, L. Caprino Department of Pharmacology - Istituto Farmacologico Serono,Rome - Italy The in vivo administration of a thymic extract (TS), in neonatal guinea pigs, in which the T-cell subpopulations express different levels of functional activity, was found, in a previous work, to differently influence the immunological responsiveness or maturation stake of the lymphoid spleen cells, according to the function studied. TS administration induced an increase in the number of antibody-producing cells, an increase in the response to mitogenie stinm/lation but no variation in the number of E-rosette forming cells. The present work illustrates the activity of TS on aged guinea pigs. TS was found, in i_nn vitro experiments to increase both the response of peripheral blood lymphocytes from aged animals to lectins, and the number of E-rosette forming cells. Furthermore the in vivo administration of TS to the aged animals resulted in a restoration of immunocompetence of T-cells as shown by the increase of the response of spleen lymphoid cells %0 sheep red blood cells and to specific T-cell antigens stimulation. I) TS = Thymostimulin - In previous papers the same product was named as TP-I
5
IMMUNOKINETICS OF A SINGLE DOSE OF THYMOPOIETIN PENTAPEPTIDE. F. La~hi Pasini~ A, Auteri~ A.L. Pasqui and T. Di Perri Department of Internal Medicine s School of Medicine~ University of Siena, 53100 Siena Italy. Previous studies have shown that 'Tin vitro" incubation with Thymopoietin pentapeptide(TPS) is able to normalize the number of E-rosette forming cells in Rheumatoid Arthritis (RA) patients when depressed. According to these premises 14 patients with classical or definite
155
RA and a low number of E-rosette forming cells were given single doses of 5 mg and 50 mg of TP5 intravenously in order to verify the "in vivo" effect of the drug. TP5 produced a dose-related sustained increase in E-rosette forming lymphocytes as shown by the decrease of thymopoietin dependent rosetting ratio. A single dose of 50 mg restored the n ~ b e r of E-rosette forming cells to normal after 12h and this effect lasted 6 to 7 days. With the administration of 5 mg of thymopoietin pentapeptide, the basal values of E-rosette forming ~;ells markedly increased after 12h and returned to the pretreatment level on the 2nd day. These immunokinetic data clarify some aspects of the clinical pharmacology of TP5 and must be considered for a rational schedule of treatment with the drug.
ISOLATION AND ASSAY OF SPECIFIC T-LYMPHOCYTE PROLIFERATION INHIBITORS (CHALONES) FROM CALF THYMUS H.R. Maurer and R.Maschler Pharmazeutisches Institut der Freien Universit~t Berlin K8nigin-Luise-StraBe 2+4, D-!O00 Berlin 33, Germany
6
Increasing evidence sugges~that the proliferation of activated T-lymphocytes is regulated not only by stimulating (e.g. T-cell growth factor), but also by inhibiting factors. Some of the latter have been found to suppress T-lymphocyte proliferation in a cell-line specific,non-toxic and reversible manner and may therefore be classified as chalones. Using agar colony assays in glass capillary tubes with truly proliferating, stimulating human T-lymphocytes and mouse granulocytes, we isolated from calf thymus acetone powder 2 ultrafiltrate fractions with chalone properties: F! in the MW range l-!OxlO- proved to ~e stable upon heating, prolonged storage and lyophilization, whereas F2 in the range !-3xlO was found to be unstable. Tween 80 and cetyltrimethylanm~onium bromide facilitated the extraction of F2 and F2. Biogel P6 chromatography of FI increased the specificity of inhibition of lymphocyte colony growt h and revealed an MW of approx. 1.400d.Upon DEAE cellulose chromatography of the active pool an inhibitor of high specific activity was eluted with 0.2 M NaCI, devoid of any granulopoiesis inhibitor. Using a washing procedure prior to the colony assay we demonstrated its reversibility of action as well as its non-toxicity. Its activity in the nM-range was found unlikely to result from spermine, but from an anionic peptide. Loss of activity upon storage due to adsorption, varying yields from 2 kg tissue batches and the low quantities ( n g - ~g) of the purified inhibitor require large scale isolation procedures which are underway. In summary, a T-lymphocyte chalone of high specific activity was isolated acting as an antigen-nonspecifie suppressor factor of T-cell proliferation dependent immune response.
ROLE OF I~{E THYTqUS IN THE ~{0DUCTION OF THE HO~,~O~£ DIFFERENTIATING ~qD ~TURIIIG T CELLS G. Renoux I ~i. Renoux and J.M. Guil1~umin Laboratoire d'Immunologie, Facult6 de M6decine, B.P. 5223, 37052 Tours Cedex, FRANCE.
The t h y m u s f r o m a f e m a l e C 5 7 B L / 6 ( B 6 ) m o u s e was i n t r a p e r i t o n e a l l y g r a f t e d t o a f e m a l e n_~/nu (B6 b a c k g r o u n d ) m o u s e w h i c h was t r e a t e d w i t h 25 m g / k g DTC, o r w i t h s a l i n e a s c o n t r o l , 15 d a y s later. S e r u m wa~ s a m p l e d 24 h l a t e r , h e a t e d a t 56°C f o r ~5 m i n , a n d t e s t e d f o r i t s c a p a c i t y t o i n d u c e T h y - 1 - c e l l s i n n_~/n~u s p l e e n c e l l s , a f t e r 7 . 5 h - i n c u b a t i o n , in a microcytotoxicity a s s a y u s i n g m o n o c l o n a l ~ - T h y - 1 a n t i b o d y . D a t a w e r e c o m p a r e d w i t h t h e e f f e c t o f ~11C on n o r m a l t h y m u s - b e a r i n g B6 m i c e . E a c h g r o u p c o m p r i z e d a t l e a s t 5 m i c e . A s y n g e n e i c t h y m u s g r a f t i n d u c e s i n t h e n_~/nu m o u s e t h e p r o d u c t i o n o f a p a r t o f t h e h o r m o n a l a c t i v i t y found in normal B6 mice. A single administration of DTC triggers in n_~/nu B6 the synthesis off a fully-active hormone. When the serum of thymus-reconstituted and DTC-treated nude mice was tested, the dose response our~e paralleled that of the serum from normal B6 mice treated with DTC, but at markedly lower thresholds of T-cell inducing capacity. A cellular system other than the thymus synthetizes under the influence of DTC the hormone specifically active on the T-cell lineage. The thymus appears as the organ which regulates and controls the production of this hormone.
7