Note: Descriptions are shown in the official language in which they were submitted.
1 32~;592
P 20 824 ~ 002/ja
March 14, 1988
Prophylaxis or a eherapy of d;sorders and d;seases ~ith;n
the gastrointestinal tract of animals and humans using
antigenic substances.
1 D E S C R I P T I_O_N
The invention refers to the prophylaxis and therapy of -
disorders and diseases within the gastrointestinal tract
of animals and humans using an ant;genic substance.
;-
Morbid disorders within the gastrointestinal tract of
animals and humans represent a world~ide serious,
economicaLLy significant problem. In human medicine, among
chronic disease, peptic ulcer disease, for instance, sho~s
a life long prevalence of about 8 - 10X ;n Europe and
North America. Thus, the total costs for peptic ulcer
pat;ents ;n U.S.A., The Netherlands, Italy and ~weden,
i,ncluding working deficit are about 1% of the respective -~
national annual expenses for health care. In 6ermany, for
;nstance, the mortality because of peptic ulcer d;sease
w~th;n the period from 1952 to 1980 has decreased only
insigificantly from 7 to 6 dea~hs per 100,000 inhab;tants
~Sonnenberg R., Fritsch, A.: Changing mortality of pept;c
ulcer disease in Germany. Gastroenterology B4, 1553
(1983)). With~n th1s per;od, ho~ever, peptic ulcer
disease, as a deadly d;sease ot middle aged and elderly
peoplo, sh~fted to disease of moinly old and high-aged ~
people. Eesides the mortality because of gastr~c and ~ -
duodenal ulcers, in women, has increased. The mortality in
mon becau~o of o duodenal ulcer remaincd constant; those
of gastr1c ulcers slightly decreased.
:-:
2 132~92
l Currently, mainly H2-antagonists are used for the
treatment of peptic ulcer disease (8auernfeind et al.,
Ulcustherapie, JAMA, H~ft 2, 135 (1986~). The principle o~
the therapie ~ith H2-antagonists is based on the reduction ;-
of the aggressive effect of ac;d uithin the
gastrointestinal tract. This therapy is e~fected ~ith a `
number of unpleasant side-effects. A significant problem
of a peptic ulcer disease still is the prophylaxis of a
recurrent ulcer because, as already mentioned~ peptic
IO ulcer d;sease is among the chronic d;seases ~ith life-long ~-
prevalence. For instanee, a long term study of three years
among 44 Centres ~;thin 13 countries ~ith a total of 1,423
patients y;elded that only 81% of patients were without
recurrence after 12 months, 73% after Z4 months and 65X
after 36 months for duodenal ulcers (DMW 111. Jg. Nr. 3,
117, (1986)). Therefore, there is the urgent necessity for
alternative drugs for the prophylaxis or treatment of
peptic ulcers ~ithout side effects ~hich especially could
be taken for a long t1me and as prophylaxis.
Diarrhoaea and peptic ulcer disease are a serious problem
not only in human medic1ne but also 1n veterinary
mod1cine, especiaLly in pig and cattle breeding as vell as
in poultry farming. It is a ~ell knoun phenomenon, for
1nstance, that p19lets after ~eaning and a change to
sem1-l;qu1d h;gh energy ratio u1ll develop ulcers ~hich
may part1ally be lethal. ~1th1n the Common Market there ~ -
are 120 to 160,000,000 p19s ~est~mated values), among
which three to four percent w1ll be atfected. Based upon
these f19ures, the loS5 of p1gs ~ll account for about 4
mill1On per year, result1ng 1n a f1nanc1al loss of up to
1, 2 b1ll1On 6erman Marks per ye~r. It ls obv1Ous that
offect1ve measures aga1n~t these d1sorders are necessary.
Noarly the same 15 true for cattle breed1ng and poultry
.-: . , .
1 farm;ng.
For the pathogeny for peptic ulcer disease, aggressive
factors during digestion, like the secretion of acid and
?epsin, seem to be of special significance (Grossman,
M.l.: Regulation of gastric ac~d secretion. ~n: Physiology
of the Gastrointestinal Tract. Ed.: L.R. Johnson. Raven
Press, New York 659-671 t1981)).
Opposite to these aggressive factors are protective
physiologic digestive mechanisms. Among these protective
mechanisms is the mucus secretion in the stomach, which -
together with the bicarbonate secretion ;s regarded as the
first layer of a mucosal barrier.
The various secretion components of the stomach, as ~ell
as the cell regeneration, are controlled by neurohormonal -
mechanisms. The hormone, wh;ch initiates the
above-mentioned aggressive digestive processes is gastrin.
It was isolated in 1964 (Gregory, R.A., Tracy, H.J.: The
constitution and properties of two gastrins extracted from
hog antral mucosa. ~ut 5, 103-117, ~1964~
Unt~l recently, one aspect concerning studies on the
stimulation of gastric funct~ons was totally not taken ~ -
into account: ~lth each meal a mucosa of the stomach also
comes ~nto contact with substances which might be
cons1dered antigenic. Although there were h;nts for
1mmunolog~cal structures in the stomach, however, there
was no idea as to their funct10ns. Recently, it could be
8ho~n, ~n exper~mental stud~es, that following preced~ng
1mmun1zat10n w;th an antigen, oral challenge ~ith sa~d
ant~gen lnduces an 1mmune reaction in the antrum of the
stomach, which lnltiates the complete ~astric cascade of
d~gest~on. This ~s ma~nly the ~ncrease of local blood flow
4 13255~2
1 in the antrum and duodenum, ~ncrease of ~ucus secretion of
the stomach as ~ell as stimulation of gastr~n releaseD The
proof of an immunological stimulation of gastric functions
offers a completely ne~ vie~ of physiolo~ and
pathophysiology of the digest;on. Th;s d~ta came from
animal stud;es in ~hich luminaL antigens, in the ~-
stomach, after preceding immunization were recognized and
induced digestive processes STeichmann, R.K~, Andress,
H.J., Gycha, S., Se;fert J., ~rendel, W.: Immunological
med;ated gastrin reLease. Gastroenterology 84,2 ~1983~
Immunofluoresence ~;croscopy revealed binding sites for
the ant;gen applied orally ~ithin the lamina propria
mucosa of immunized animals. Furthermore, there ~as an
increase in the number of cells expressing immune
15 associated antigens on their surface (so-called
Ia-antigens) in the antrum, following antigen
administration. Using double immunofluoresence microscopy
it could be demonstrated that the binding sites for the
antigen seems to be located on Ia-antigen expressing
20 cells.
furthermore, there was an increase in plasma in mast
cells, in the lamina propr;a mucosa of ;mmunized anlmals,
after antigen administration and an increase in the mast
cell degranulation, as ~ell as an increase in
25 ~ntraepithelial lymphoid cells. ~Teichmann, R.K., Andress,
H.J., Lieb~ch, H., Seifert, J., Brendel, ~.: Die Bedeutung
immunkompetenter Zellen 1m Antrum bei der Stimulation von
Verdauungsprozessen. Langenbecks Arch. Chir. Suppl. 151,
t~984)).
Thus, during immunologically mediated stimulation of
ga8tric digestive proce3ses, bes~des a~ress~ve factors
like acid also zytoprotective mechan~sms l~ke increase of
132~2
1 blood flo~ and mucus secretion, are init~ated.
Based upon these resul~s further experiments ~ere carried
out concerning mediators of the immunological sti~ulation
of gastric functions using the model of isolated
antrum-muscular perfusion in dogs. Gastrin and
prostaglandine E2 and F2 determinations ~ere performed in
an intravenoùs effluent.
The supernatant of fluoresic duct Lymph, collected after
antigen administration in immunized animals, stimulated
gastrin release in ;solated antrum perfusion. This
activity was missing in the lymph of non-immunized animals
when the same antigen was administrated into the stomach,
tTeichmann, R.K., Pratschke, E., Grab, J., Tutert, J.,
Enders, G., Brendel, W.: Gastrinstimulierende Wirkung von
Ductus-thoracicus-Lymphe nach gastrointestinaler ~ ~-
~mmunreaktion. Langenbecks Arch. Chir. Supp(. 271
t1985)).
Thus, it has been shown that soluble mediators are
responsible for the antigen induced gastrin reLease.
The mucosa mast cell products Histamin and Leukotrien C4,
being released dur~ng the immunological react;ons,
significantly inhibit antral gastrin release. Gastrin
it~elf stimulates the release of Histamin so that these
results, for the first time, show local antral negative
feed-back mechanism bet~een the G-cell and the mast cell
tPratschke, E., Teichmann, R.K., Grab, J., Tutert, E.,
Brendel, W.: Der Einflun von Mastzellprodukten auf die
Gastr1nfreisetzung. Langenbecks Arch. Chir. Suppl., 295,
t1985)).
Simultaneous to tho inhib1tion of gastrin release by -~ -
" ' ''
6 13255~2
l Histamin and Leukotrien C4, thece ;s a r~lease of
endogenous prostaglandine E2 and F2. In the model of
isolated antrum perfusion it could be sho~n that
prostaglandine E2 inhibits the gastrin release. This
~ight indicate that the inhib;tion of ~strin, by mucosa
mast cell products, may be ~ediated by endogenously
released prostaglandines (Pratschke, E., Teichmann, R.K.,
Grab, J., Enders, G., Brendel, W.: Endogenes Prostaglandin
E2 als Regulator der Gastrinfreisetzung. In:
Kongrenbericht der 26. Jahrestagung der osterr.
Gesellschaft fur Chirurgie. Hrsg.: F. Helmer, E~ Horcher,
Styria-Verlag, 6raz, 131, (~985)~.
During the process of antigen recognition involving
antigen presenting cells lymphokines Interleukin 2 and
Gamma inter~eron are released. Both stimulate gastrin.
Thus, for the first time, stimulation of a gastrin by
mediators of immuno competent cells was sho~n (Pratschke,
E., Teichmann, R.K., Grab, J~, Hammer, C. Brendel, ~
Gastrinfreisetzung durch Mediatoren lmmunkompetenter
ZelLen. Langenbecks Arch. Chir. Suppl., 261 t1986)).
Immunofluoresence microscopic studies of the antrum of the
perfusion with Interleukin 2 and Gamma interferon, using -
monoclonal an~ibodies aga;nst class 2-antigens of the MHC
complex, revealed further that, after the perfus10n ~ith
antigenic structure of DLA Class 2 ~Dog-Leukocyte
ant~gens), same are expressed on epithelial cells,
macrophages, probably also on intraepithelial
T-Lymphocyte. Th~re was no expression of those t~o
ant1gens after perfus~on ~ith Interlcukin 2.
Therefore, Interleuk1n 2, but also Prostagland1nes and
other substances, may serve as mediators of this
~mmunolog1cal r~action.
.
-- 1325592
1 Thus, the above-mentioned antigen induced reactions are
discussed for the development of ulcers and may also be of
significance for the total gastro;ntestinal tract.
Whenever antigen induced increase of the antrum blood
flo~, gastrin and mucus secretion is missing, or disturbed
locally, the resulting acid may not be sufficiently
buffered and transported back in case of lack of increase ~-
of blood flo~, resulting in penetration of the acid into
the mucosa and possibly inducing local ulcers there.
Investigations on the immunological stimulation of gastric -
reactions also showed that after ;mmunological stimulation
with a synthetic antigen, besides the antigens specific in
v;ew of the gastrin release, and the strongest stimulator
for the gastric acid secretion, in the sense of an ~-
increase of aggressive digestive processes, also an
increase of antral secretion of Somatostatin ~as noted
which has a protective effect by inhibition of acid and `
pepsinogen secretion, tKr3ml;ng, H.-J., Teichmann R.K.,
Merkle, R., Enders, G., Brendel, W.: Gastrale Protektion -~
durch immunologisch induzierte Somatostatinfreisetzung,
153. Tagung Verein;gung Niederrhein;sch-Westf~lischer
Chirurgen, K~ln, 9.-11. Okt. 1986). -
~, ., . -
This data results in a completely new far reaching, and so
far not recognized, possibil;ty to specifically influence
digestive processing as a protective mechanism concerning
all disorders and diseases within the gastrointestinal
tract. In human med;cine, d~seases like eros~ons, peptic
ulcors and, last but not least, cancer are of greatest
interests. In veter1nar~ medicine interest ls mainly in -~
the critical trans~tlon phase of young animals follouing
~eaning and subsequent contact u1th high energy food
because of oc¢urrence of erosions and peptic ulcers
:'',' ' :' '
, . .:
. .
8 132~92
1 during this phase.
There is a protective induc~ion of functions gf t~e
~a~da~ t~R~ &tonb~dr~8~i~hs ~YI~ O~fit~
functions and protective physiologic di3est~ve processes,
~hich may be used in a broades~ sense; th;s is the use of
an ant;genic substance for the prophylaxis and therapy of
disorders and diseases cf the gastrointestinal tract of
an;mals and humans induc;ng protect;ve phys;ological
d;gest;ve processes after an oral chaLlenge of the animal ~ -
or human ~ith the antigenic substance following preceding
immunization~
~hen applying an antigenic substance, as outlined by the -
invention, the stimulated gastr;c functions may ~ork as
follows:
After immunization of the stomach ~;th an antigen wh;ch is
present in the food the subsequent contact of this
antigen, ;n the food, ~ith the gastric mucosa, the antigen
~ill be recognized by the so-called antigen presenting
cells, for instance, ~acrophages and presented to the
T-lymphocytes by the help of cLass 2 antigens. An
;ndicat;on for a lympocyte mediator ;mmunolog;cal ~-
stimulation of the above-mentioned gastric functions ~as
demonstrated in an exper;ment ;n ~hich ;mmunized dogs ~ere
irradiated by an 80 Rad of an ultra-hard X-ray resulting
tn an inhibition of the immunologically st;mulation of
gastrin release based upon destruction of lymphocytes. The
capability of the gastrin cell to be sti~ulated, ho~ever,
is not effected by the irradiation.
.:
Follo~ing the contact of the antigen ~ith the stomach,
be1ng immunized ag outlined above, there is a type of
132~2
I cascade of release of Interleukin 1 and st;~ulat10n of
T-Helper-Lymphocytes. The Helper cells theuselves ~iLl
reLease Interleukin 2 and Gamma interferon, t~o
lymophokines ~h;ch ~ere sho~n to be able to release
gastr;n. A s;gnificant protective physiological mechanism,
namely the increase in mucosa blood flo~ representing one
of several steps of the cascade of protective digestive
processes, may be caused by mediators of ~ast cells,
especially Histamine. Besides the expression of -~
class-2-antigens on antigen presenting cells of the
antrum, Gamma ;nterferon also induced the e~press;on of
these antigens on epithel cells. Possibly th;s additional
recruitment of epithel ce~ls represents a potentiation of
antigen recognition. The vagus nerve interferes with this
antigen recognition system, in the sense of
immunomodulation. The mentioned inter-relations are sho~n
in Figure 1.
The ulcer protective effect of antigen spec;fic ~-
stimulation of d;gestion has been demonstrated in an
an;mal model. Using the model of the "SHAY"-Ulcer (Shay.
H., Komarov, S.A., Fels, S.S., Mekanze, D., Gruenstein,
M., Sip~et, H.: A s;mple method for the uniform production
of gastr;c ulceration ;n the rat. Gastroenterology 5, 43,
t1945)), the influence of antigen stimulated regulation of
the digestion was invest;gated. Male ~istar-Rats ~ere
immuni~ed system;cally ~th a Hapten. After~ards, the
Hapten ~as administered into the stomach and as control
sodium chlor;de was g1ven. In further control studies, the -
Hapten or sod~um chlor;de, respectively ~as also
admin;stered 1n non-;mmun~zed animals. These rats being
treated in such a way were invest1~ated macroscopically
and m~croscopically to check gastric lesions after the~r
death but not later than t8 hours. There was a
signif;cantly less express;on or number of perforations
'10
i32~92
1 and large ~all necroses tn the core ventr~culous in the
group of animals ~hich had been immuni~ed ~th the Hapten
(Figure 3.)~
Thus, us;ng the model of the "SHAY-Ulcer~ the protective
effect of an antigen specif~c st;~ulation of the d;~estion
could be demonstrated. Although there is an ulcerogenic
effect by release of gastrin and, consecutively of acid,
as an aggress~ve factor, simultaneously there is also
stimulation of protective mechanisms, for instance, an
;ncrease in blood flow, mucus secretion, possibly through
release of Somatostatin, Prostaglandines and other
mediators which finally prevent the occurance of severe
secret;ons of the gastric mucosa or gastric ~all. This -
ulcer protective effect of an antigen induced regulation
of the digestion, as shown here, may teleolog;cally well
be regarded as a "logical" regulation ~ithin physiologisal
processes, as it results simultaneously in a stimulation
of the digestion and a reduction of the risk to develop an
ulcer. Thus, the develoment of pept~c ulcer may well be
understood as a local failure of the stomach to be ready
for antigenic r~actions beside other kno~n causes. The
stimulation of ~astr;c ~mmunological mechanisms by
immun1zat~on ~ith antigens, therefore, results in a
completely ne~ possibility for the prophylaxis and therapy
of disorders and diseases ~1th;n the gastrin intest~nal
tract of animals and humans offering broadest ~nd;cations.
The essential, other advantage of use of antigenic
substances, for th1s purpose, is based on the fact that
the ant~ens belng used are b10log~cal substances ~ith
uh;ch the mammal a~ vell as the human being comes into
contact naturally and, thus, there ~s no aggrossivo effect
on physcolog1cal processes but, moreover, an induction of
~ logical protective physiological process in a natural
manner and, thus, same is ~ithout s~de effects.
:
~1 1 325~2
1 In respect of the mentioned mechanisms of the
immunological stimulation of the digestion, it is, of
course, of special advantage ~o use such subst~nces as
antigens which later w;ll also be included ~ith;n food.
Follo~ing immunization of the animal or the human ~ith the
antigenic substance a level of factors, uithin protective
effect, ~ill be increased in sueh a ~ay that disorders or
d;seases of the gastrointestinal tract ~ill be prevented
or treated. Thus, plan~ proteins, preferrably from maize
or soya bean, but also animal prote;ns, are especialLy
suitable for the immun;zation because these proteins are ~
often found in food or food products of animals or humans. -~-
. ,
Whey protein can, for example, be used as an amimal
protein. It became apparent that the prophylactic
influencing of gastral mucosal changes (erosions), e.g. in -
pigs, wh;ch were additionally administered ~hey protein
via the drinking water during the entire fattening period
until slaughtering of the pi~s after a fattening of 10
weeks, entailed that in particular ser;ous changes of the
~0 mucous membrane in the animals treated, as described
above, occurred less frequently by 70X than in a control
group. The ~hey protein should preferably be present in a
3 to 9X ~hey protein solution and especially preferably in -~
a 6% ~hey protein solution.
:
Human proteins, ~hich are components of blood or ~hich may
be found 1n serum or other blood derivates, for instance,
hemoglobin are suitable to be used as antigenie substances
~n the sense of the invention.
Al~o other ~ntigens, ~hich do not necessarily correspond
uith substences bein~ present ~n the ingested food, may be
su~tablo for the induction of the immunologic~l situation
12 132~92
1 ~?h;ch is respons~ble for the above-mentioned induction of
mechanisms. For instance, in case of immunization ~ith
proteins derived from the envelopes of virus, an
immunization may be possible, ~?hich ~ ork in the sense
of the above-described s~imulation of the digestion, but
also may cause an ;mmunization against virus.
Same is true for antigenic substances ~hich are present in
microorganisms with which the gastrointestinal tract may
come into contact in one ~ay or the other. The stomach
might induce the above-mentioned protective processes as
well. Specially suitable seem to be lipopoLysaccharides of
Escherichia coli, a natural commensal bacteria of some
mammals which is al~ays found in the food as contamination
and, thus, seems to be suitable. ~-
The use of an antigenic substance being present as a
synthetically-produced protein, preferably as a short
peptide which conta;ns ant;gen;c determinants for the
induction of an immune response, is especially preferred
because then a special and directly applicable prophylaxis
and therapy ~ill be possible.
A well suitable substance for the induction of the immune
sltuation to induce the protective mechanisms are Haptens,
preferably nitrophenyl acetic acid tNIP). This
substance has shown its special usefulness in the
experiment to induce the immunization, ~hich prevents the
development of mucosal lesions after intraluminal
challenge of an 3nt~gen specif~c stimulation of the
stomach. Espec~aLLy ~ith this substance, sn
immunologically mediated ulcer protect~on vss sho~n for
the f~rst time.
Preferably, the above-mentioned nitrophenyl acetic acid
l3 1 32 55~ 2
1 is used by coupling same to a carrier protein, namely
ovalbumin. -
As outl;ned ;n ~he description above, immune modulatory - ~
substances are in the equilibrium state bet~een the ~ -
aggress;ve and protect;ve digestive mechanisms. The
addition of immune modulatory substances together uith the ~ ~
antigenic substances may, therefore, cause an increased --~ -
effect of the induced protective digestive mechanisms. --~-
The use of combinations of one or several of the
above-mentioned substances may also result in an improved
prophylaxis or therapy.
- ~.,
The pharmaceutical confectioning of one or several of the~ ~-
above-mentioned antigenic substances, perhaps by adding of
immune modulators, may be performed for the enteral as
well as for the parenteral appl;cation. Concerning the
enteral confectionlng, aerosols, being applied to the nose
or the bronchial system, are preferably used. The
confectioning of the parenteral application of antigenic
substances preferably concerns substances to be used for
;ntramuscular and/or sub-cutanous applications.
In the veterinary medicine, a preferred use of the
antigen1c substances is to 1mmunize piglets with the
antigenic substance about tuo ~eeks after birth. As has
already been mentioned above, piglets suffer from peptic `
ulcers after the weaning and thé change to semi-liquids
energy-food. Thoy may even die from the~e ulcers. Treating
these piglets in this cr~t~cal phase ~1th anti~ens which
later ~ill be present in the food prevents, or at least, ``
roducos damages. The proteins being u~ed as antigenic
substances are biologic~l substances ~hich, after
appl1cat~on, will be metabolized completely ~ithout
13255~2
1 residue or being excreted in only small amounts.
An espec;ally preferred treatment of the pigle~s is to
immunize them intramuscularly, preferably the first
injection be;ng about t~o ~eeks after birth; a second
injection about four to five Yeeks after birth and a third
injection shortly before changing of food. In respect of
their practicability, the animals could then be immunized
when they receive iron-injections dur;ng breeding. The
antigenic substances may be packed in a kit ~hich already
contains iron. - -
When the piglets receive the food ~hich contains
said proteins, with ~h;ch the an;mals were immunized,
the ant;gen may also be given to the drinking water
in order to further induce and maintain the protection
of the gastric mucosa by the descr;bed ;mmunological
mechan;sms.
ln human medicine, the use of antigen;c substances ;s
preferably su;table t~ prevent or reduce erosions, peptic
ulcers or cancer in the human gastrointestinal tract. The
lncreased production of mucos may prevent the mucosa of
oncogenic substances. Furthermore, oncogenic substances,
~h~ch are known for the organism, may become inaffected by
the indùct10n of the digestion.
Also, there ~s a ne~ preferred and broad spectrum of
indications in the prophylaxis concerning diarrhoea in
travellers who come 1nto contact ~ith food products during
the1r tr1p ~h1th ore, so far, unknown by the1r
gastrointestinal tract, this means that the
g~stro1ntestinal tract, so far, had not been 1n con~act
~ith such food products. The lack of induct10n of
d19est10n may cause passage of protein 1nto deeper parts
1325~2
l of the gut with subsequent poss~ble overgrowth of
bacter;a.
It is also a well known problem in the breed1ng of - -
domestic ani~a~s that folLowing transportation of the
do~estic animals to places at which the breeding will be --
continued after the first ~eek of life, the changes ~n the
environ~ent and food may cause significant diarrhoea
which, again, may be the cause of other bacterial or viral
diseases of the whole gastrointenstinal tract. ~ -
Im~un;zat;on of these animals with antigenic substances, -
be;ng known to be present later in the food, may prevent
these disorders and d;seases.
One poss;ble use of an antigenic substance is to sensitize
piglets parenterally as of the third week at intervals of
fourteen days with 2 ml each of the 6% whey protein
solution w;thout the addition of adjuvant that is to say
on the whole with 4 ml. After weaning of the piglets ;n
the second week and gradual change over from p;g breed;ng
feed to coarse grain fattening fodder the piglets ~ere
brought into another pig sty and kept in groups ;n loose
housing boxes. From this time on approximately as of the
tenth week, all p19s were exclusively fed ~ith very finely
ground fatten;ng feed. In literature the administration of
such feed ;s ~ndicated, bes~des stress factors, as the
decisive cause of the formation of mucosal changes, such
as hyperkeratoses, erosions or ulcera in the gastric
mucous membrane of the pig.
Th- p19s were administered 3 to 6% whey protein with the
dr1nking ~ater during the entire fattening period until
the ~aughter1ng of the p~gs, after a fattening of ten
w e o k g . .:: '
16 ~32~2
1 In animals which have been treated in this ~ay changes of
the mucosa-like erosions appeared much less frequently.
In the same ~ay as the ant;genic substances have been used
for the treatment of piglets, calves may be immunized
parenterally and/or enterally in their critical phases of
life. It is preferred to give intramuscular injec~ions of
the antigen in calves ~ithin about the first four ~eeks
after birth, a second injection at about the sixth ~eek
and a third injec~ion in about the eighth to tenth ~eek
when changing to çalf food.
Within the first ~eeks of breeding, the calves receive
miLk and calf fo~d and, generally, energy food from the
eighth to tenth week. From the time of changing to the
h7gh-energy foodO the antigenic substance is added,
preferab(y to the food and/or drinking water to use
and/or maintain the level for the protection of the
intestinal tract ~y the described immunological
mechanisms.
The immunization is preferably performed also ~ith adult ~-
rum~nents. Bulls and co~s increas~ngly suffer from gastric `~
ulcers of d~fferent stages during the high lactation
per~od. These ulcers are also especially considered as the `~
cause of subsequent infectlous diseases of the ~hole
gastrointestinal tract ~n adult cattle. Enteral or
parenteral treatment of animals ~ith the antigenic
substances may prevent these damages.
Anothor field of using the antigenic substances is the
treatment of domestic poultry, for ~nstance, chicken,
turkoys, geèse, ducks and turkey-cocks. The ma~or sources
of food, for the breed~ng of domestic poultry, are grain
granules, fish flour, ~oya and grlnded peanuts~ To induce
:, ,
132~5~2
l the unisat;on, in respect of the above-mentioned food
substances, a parenteral injection of the antigenic
substance ~ill be performed on the first day after
hatching because of pracical reasons. Later,
immunizations~for maintenance of a preceding immunization
may be performed by application of the antigen via the
drinking ~ater ~ithin the early days or ~eeks of life.
. .
The current invention ~ill no~ be described, in more
detail, ;n the follo~ing Figures and Examples.
It is shown in
Fi9Y~-1:
The regulation of antigen stimulated gastrin release.
Luminal antigen is recogn;zed by ant;gen presenting cells -
and may stimulate mast cells as ~eLl as T-lymphocytes
among others. The T-cell products Gamma interferon and ~
In~erleukin 2 cause release of gastrin which~ in turn, -
stimulates Histamin. The mast cell products Histamin,
Leukotrien C4 and Prostaglandine E2 again inhibit the
gastr~n release ~from negat~ve feedback). The vagus nerve
may also 1nfluence the T-coll systems and the antigen
presont~ng cells.
.
i9
Ulcer 1ndex us1ng the model of the alcohol ulcer of the
rat.
' ~ '
Rats being immunized ~th NIP sho~ a s~gn1ficantly lD~er
.
1325~92
l ulcer index follo~ing ;ntragastric application of the
antigen.
Fi~Lure_3:
.
Percentage of animals with lesions, induced by the
SHAY-Ulcer modeL in controls and in animals immunized ~ith
N$P-ovalbumin.
The intragastr;c appl;cation of NIP-human Gamma-globin
(NIP-HGG) results in a significantly lower expression of
peptic ulcers in immunized animals.
i~Ur5t_4:
Quantitative changes of the evaluation scales (O and ~
after a pathologicaL-anatomic evaluation of the gastric
mucous membrane of pigs in the area of pars
proventricularis after a 10 ~eek feeding of very finely
ground feed rations ~X).
"
':' . .
Tho purpose of the described examples ~as to investigate ~- -
the effect of the immunolog~cal processes on the other
1nduct;on concerning stimulation or protection~
Male W1star-Rats t250-3509) ~ere 1mmunized systemically
w1th NIP-OA ~Nitrophenyl acetic acid-Ovalbumin). To
induce ulcers, t ml of absolute alcohol ~as administered
into the stomach under ether anesthesia. Fifteen m1nutes
pr10r to tho alcohol application, one group tI) of the
~ 19 1~25~92
l animals (n=12), received the antigen NIP tcoupled to HG6
as carrier-protein), Group II (n=12), the carrier protein
HGG alone. Non-immunized animals, six having rece;ved
NIP-HGG tIII), a further six HGG ~IV), proved as further
controls. An hDur after the administration of alcohoL, the
lesions were determined after the animal ~as killed and
the stomach removed. The les;ons ~ere meaSUreLi
macroscopicalLy by estimatation of the length of the
hemorrhagic lesions.
Animals having been immunised systemicallY ~ith NIP
developed significantly less mucosal lesions after oral
NIP-application and subsequent administration of alcohol
(p = 0,02) than all control groups.
I~ble _1
Immunisation Intragastric Length of
Administration of hemorrhagic
Lesions
_______________________________________________ __________
I ~n=12) NIP NIP 24 p=0,02
II tn=12) NIP HGG 57
III tn=6) non-imm. NIP 72
IV ~n=6) non-imm. HGG 78
__________________________________________________________
The intraluminal antigen specific stimulation of the
stomach in immun;sed Wistar-Rats significantly reduced the
development of mucosal lesions. Therefore, for the first
25 time, ~n immunologically mediated ulcer protection is
sho~n tFigure 2).
20 ~32~2
E,~ anD lg_ 2
The immunization of male Wistar-Rats (250-3509) was
performed using NIP-OA (Nitrophenyl acetic
acid-Ovalbumin). Pylorus ligated rats (SHAY-Ulcer) served
as ulcer model. Follo~ing ligation of the pylorus under
chloralhydrate anesthesia, the immunized rats rece;ved the
antigen NIP (coupled to human gamma globulin) directly -
into the stomach (tesS group). The control animals
rece;ved only the carrier protein HGG. Another control -
group consisted of sham operated animals (without pylorus
ligation). Eighteen hours after the operation, the
changes ~n the stomach wall were investigated
macroscopically and microscopically. The lesions were
defined as follows:
:.
Perforat;ons, confluent transmural ulcerus and all
necroses of more than 0,5 cm in diameter.
An;mals which ~ere systemically immunized with NlP showed ; `
significantly less lesions after pylorus ligation and
intragastric application of NIP-HGG ~p = 0,05) than
controls which had received HGG. Sham-operated rats
showed no mucosal lesions.
~-....
.
.'..' ;''. '.
~, .. .
132~92
1 ~abl~_2 ~ -
_______________ ____ . .~ .
NIP-specifically Intragastric Proport;on of
;mmun;sed animaLs Applisation of Animals shoYing
Lesions
____--________________------------------------ :
5 Test group tn=24) NIP-HGG 7/24 -~
Control group (n=23) HGG ~4/23 -
Sham-operated ~n=6 NIP-HGG resp.HGG 0/6 resp. 0/6 ~-
resp.6) -
__________________________________________________________ :~
The antigen specific stimulation of gastric funct;ons
result in a slgnificant reduction of wall lesions in a
defined ulcer model tPylorus ligation) (Figure 3.).
~Q1~_3
~r.~C~.gYr~._Qf_~ Y~ -QQ-Q~--pi~~
Supposedly the antibody production in piglets starts from
lS the second week and regarding the practicability, the
plglets are immunized and receive iron injections during
their breeding by the breeder.
A blologlc protein or a synthetic antigenic immune
stlmulating substance ~ll be used as antigen. It ~ill be
~etabolized without residues following digestion by the
piglets or it uill be excreted, as such, in only small
,' ' '
. '~ '
132~592
l amounts.
For the present immunization of the piglets whey proteins,
namely as a 6% whey protein solution, ~ere administered to
the piglets without the addition of an adjuvant or during
the later course of immunization as a 3 to 6X addition to
the drinking water~
Thirty pigs ~ere taken as a test group and thirty pigs as
controL animals for an examination of the prophylactic
effect of the ~hey proteins administered as antigens
regarding gastral mucosaL changes. Dur;ng the test f;ve
pigs of the test group and t~o of the controL animals died
as piglets or during change-over.
The test animals were pretreated according to the
follo~;ng scheme. Piglets were sensitized parenterally as
of the third week at intervals of 14 days ~ith 2 ml each
of a 6X whey protein solut;on ~;thout the addition of
adjuvant that ;s to say on the ~hole with 4 ml.
After weaning of the piglets in the second ~eek and
gradual change over from pig breeding feed to coarse grain
fattening fodder both the test and the control ani~als
were brought ~nto another p19 stay and kept in groups in
loose hous~ng boxes.
From th~s time on, approx~mately as of the 10th ~eek, all
pigs ~ere exclus;vely fed ~1th very finely ground
fatten1ng feed. In literature the administration of such
a teed is ;ndicated besides stress factors as the decisive
cause of the format~on of mucosal changes, such as
hyperkeratoses, eros10ns or ulcera in the gastric mucous
membrane of the p~
~ 23 i32~5~2
l Both groups of animals were administered 3 to 6X whey
protein ~ith the drinking ~ater dur;ng the ent;re
fattening period till the slaughtering of the pigs after a
fattening period of 10 weeks.
.. .- -
The changes of the gastric mucous membrane of pigs in the
area of pars proventricularis W35 evaluated according to
pathological-anatomic criteria.
Table 3 shows the ~requency of the evaluation scales tO to -
+++) after a 10 weeks feeding of very finely ground feed
rations and their percentages (X).
.
Lag-l--3
O + ++ +++
6 6 8 8 control group
21,4% 21,4X 28,6X 28,6% n = 28
1û 7 6 2
40X 28% 24Z 8X test group
n = 25.
In Figure 4 it is graphically represented that in the
animals treated ~ith whey protein t~ice as much animals
had an intact mucous membrane than ~n the controls (the
t~o left columns in Figure 4 ~ith the designation "0"
which is usod for an intact mucous membrane). The two
right columns of F1gure 4 reveal on the other hand that
ser~ous changes of the mucous membrane, e.g. erosions,
~h1ch are des~gnated ~ith "~" occurred by 70X less
frequently than in the control.
, ...
24 1325~2
1 The exact figures of the macroscopic evaluation of the
gastric mucous membranes of the experimental animals as
they are reveaLed by table 3 sho~ that lo~ grade, flat
hyperkeratoses (designated ~ith "+") or moderate
hyperkeratoses with pointed serrations (designated ~ith
"++") occurred ~ith the same frequency in both groups.
However, the changes ~ith "+~", i.e. the erosions, are
dec;sive, ~hose prevention in this test ~ith pigs ~as
possible still more clearly than in the experiments
described in examples 1 and 2. `
E~ampl~_~
P~rfoc~c~ of lm~unisatiQn_of_CattLe
As antigen, a protein is used which ;s not present in the
calf food, especially not in the milk replacer with which
the calves are fed after birth. However, the antigen must
be present ~n the high-energy food ~hich is given to the
calves in about the eighth to tenth week after birth. The
ulcerative lesions preferably develop during the change to
h~gh-energy food, usually at an age of four to ten weeks. -~
These disorders are in connection ~ith the mentioned lack
of immunization of animals regarding the proteins being ~ -~
present in the h1sh-energy food. The main constituents of
the high-energy food for rum1nents are mainly corn, ~heat,
soya, barley, grassilage and hay. These constituents may
change according to the degree of ~ntensive breeting.
The f1rst ~ntramuscular ~n3ection of the used antigenic
~ubstance ~ll be given in the first four ~eeks after the
b~rth of the calf. A second injection ~;ll follo~ in the
s1xth ~eek and a third in3ection at the time when
high-energy food will be given in the eighth to tenth ~
: " :' -
'- . ' "
.
,5 1325~2 :
1 ~eek. In the first week of breeding the calves receive
milk and starter diet~ They wi~L rece;ve high-energy food
about the eighth to tenth ~eek. Then~ at the time of
changing to the high-energy food, the antigenic substance
will be added to the food and/or drink;ng ~ater to use
or establish the immunological protection of the
gastro;ntestinal tract by the above-described
immunological mechanism.
The dose o~ the antigenic substance should be about 0,19
test substance per kg body~eight by intramuscular
application and will be repeated two to three times.
To induce the protective mechanisms, the antigenic
substance will then be added to the drinking water ;n a
dose of up to 19 per 100 ml water.
Performing this procedure, the ulcerative lesions in the
gastro;ntestinal tract of the calves ~ill be prevented or
reduced.
:,
Accord;ng to the above-mentioned handling of the cattle,
they will be much better protected against changes of the
climate, the food and transports ("Crowding Disease"),
especially ~ithin the f;rst ~eeks after b;rth. The
frequently-occurrSng d;sorders or diseases of the
gastrointestinal tract, during these environmental
changes, especially the diarrhoea, may ~ell be prevented
by the new mechanism. Diarrhoea can be the cause of other
bacterial or viral diseases in the gastrointest;nal tract. - -
Concerning the use of antigenic substances in adult
ruminants, the required antigenic substances ~ill be
appl~ed in bulls and cows during the per~od of intensive
feeding and high lactation. Thus, also in adult animals
26 132~92
1 the more common gastric ulcers, of various stages~ may be
prevented or reduced.
Exa~J21~_5
Performance of immunization of poultry
SimiLar to the ;mmun;zation of pigs and cattle, domestic
poultry, for instance, chickens, turkeys, geese, ducks or
turkey-cocks, ~ill be immunized with antigenic substances
in the above-mentioned way. The antigen;c substances ~
consist of grain, fish flour, soya or peanuts according to
the major sources of food during the later breeding.
The parenteral inject;on of the antigenic substance is
performed on the f;rst day after hatching due to practical
reasons. This first immunization ~;ll be intens;f;ed and
estabL~shed by later application of the ant;genic
substance by putting the antigenic substance into the
drinking ~ater in an entral form abou~ one week after
the hatch;ng. ;~
''" ' ~. '.. .
-
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...
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