IE66559B1 - Prophylaxis or a therapy of disorders and diseases within the gastrointestinal tract of animals and humans using antigenic substances - Google Patents

Abstract

Disorders and diseases in human and animal digestive systems, for example diarrhoea, ulcerative disorders and carcinomas, are a serious, economically important problem worldwide. It was possible to show that immunological mechanisms exhibit a protective effect in the gastrointestinal tract of animals and humans. Antigens contained in the food induce the said protective mechanisms if the body has previously been sensitised to this antigen. The use of antigenic substances in the prophylaxis and therapy of ulcerative disorders in humans is preferred. The antigenic substances according to the invention are furthermore particularly highly suitable in the raising of young cattle, which are subjected to a change of food in the first weeks of life.

Description

BE S € JK £ F T £ 0 M The invention refers to th® prophylaxis and therapy of disorders and diseases within the gastrointestinal tract of animals and humans using an antigenic substance» Morbid disorders within the gastrointestinal tract of animals and humans represent a worldwide serious,, economically significant problem. In human medicine, among chronic disease, peptic ulcer disease, for instance, shows a life long prevalence of about 8 - 10^ in Europe and Month America., Thus, the total costs for peptic ulcer patients in O.S.A., The Netherlands, Italy and Sweden, including working deficit are about 1% of the respective national annual expenses for health cars. In Germany, for instance, the mortality because of peptic ulcer disease within th® period from 1952 to 1980 has decreased only insigificantly from 7 to ό deaths per 100,000 inhabitants (Sonnenberg R-, Fritsch, A.: Changing mortality of peptic ulcer disease in Germany. Gastroenterology 84, 1553 <193555» aithio this period, however, peptic ulcer disease, as a deadly disease of middle aged and elderly people, shifted to disease of mainly old and high-aged people,. Besides the mortality because of gastric and duodenal ulcers, in women, has increased. The mortality in men because of a duodenal ulcer remained constant; those of gastric ulcers slightly decreased.

Currently,, mainly ^-antagonists are used for the treatment of peptic ulcer disease (Bauernfeind et al™, Ulcustherapie, JAMA, Heft 2, 135 (198635.. The principle of the therapie with H2-antagonists is based on the reduction of the aggressive effect of acid within the gastrointestinal tract- This therapy is effected with 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 ulcer disease is among the chronic diseases with life-long prevalence- For instance, a long term study of three years among 44 Centres within 13 countries with a total of 1,423 patients yielded that only 81% of patients were without recurrence after 12 months, 73% after 24 months and 65% after 36 months for- duodenal ulcers (OHM 111- Jig- Nr® 3, 117, (19863)- Therefore, there is the urgent necessity for alternative drugs for the prophylaxis or treatment of peptic ulcers without side effects which especially could be taken for a long time and as prophylaxis20 Oiarrhoaea and peptic ulcer disease are a serious problem not only in human medicine but also in veterinary medicine, especially in pig and cattle breeding as well as in poultry farming- It is a well known phenomenon, for instance, that piglets after weaning and a change to serai-Iiquid high energy ratio will develop ulcers which may partially be lethal- felithin the Common Harket there are 120 to 160,000,000 pigs (estimated values), among which three to four percent will be affected. Based upon these figures, the loss of pigs will account for about 4 million per year, resulting in a financial loss of up to 1, 2 billion German Glarks per year- It is obvious that effective measures 'against these disorders are necessary. Nearly the same is true for cattle breeding and poultry V $ fa run ng® ί! for the pathogeny for peptic ulcer disease,, aggressive factors during digestion, like the secretion of acid and pepsin, seem to be of special significance (Grossman, H-I.: Regulation of gastric acid secretion. Ins Physiology of the Gastrointestinal Tract® Ed®: t®R® Johnson® Raven Press,,. Neu York 659—671! ¢1981)), 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 is regarded as the first layer of a mucosal barrier® The various secretion components of the stomach, as well as the cell regeneration,, are controlled by neurohormonal mechanisms. The hormone, which initiates the above-mentioned aggressive digestive processes is gastrin® It was isolated in 1964 (Gregory, R,A®, Tracy, H.J.x The constitution and properties of two gastrins extracted from hog antral mucosa® Gut 5, 103-117, ¢1964)), Until recently, one aspect concerning studies on the stimulation of gastric functions was totally not taken into account: with each meal a mucosa of the stomach also comes into contact with substances which might foe considered antigenic- Although there were hints for immunological structures in the stomach, however, there was no ides as to their functions® Recently, it could be shown, in experimental studies, that following preceding immunization with an antigen, oral challenge with said antigen induces an immune reaction in the antrum of the stomach, which initiates the complete gastric cascade of digestion. This is mainly the increase of local blood flow in the antrum and duodenum, increase of mucus secretion of the stomach as well as stimulation of gastrin release» The proof of an immunological stimulation of gastric functions offers a completely new view of physiology and pathophysiology of the digestion» This data cam® from animal studies in which luminal antigens, in the stomach, after preceding immunisation were recognized and induced digestive processes (Teichmann, R-Κ», Andress, H-J-, Gycha, S-, Seifert 4-, Srendel, H.: Immunological mediated gastrin release» Gastroenterology 84,2 (198355» Immunofluoresence microscopy revealed binding sites for the antigen applied orally uithin the lamina propria mucosa of immunized animals» Furthermore, there was an increase in the number of cells expressing immune associated antigens on their surface iso-called la-antigens) in the ant rum, following anti gen administration» Using double immunofluoresence microscopy it could be demonstrated that the binding sites for the antigen seems to be located on la-antigen expressing cells.

Furthermore, there was an increase in plasma in mast cells, in the lamina propria mucosa of immunised animals, after antigen administration and an increase in the mast cell degranulation, as well as an increase in intraepithelial lymphoid cells. CTeichmann, 2UK-, ftndress, H.4., Liebich, H-, Seifert, J., Brendel, M-: Die Sedeutung immunkompetenter Zellen im Antrum bei der Stimulation von Herdauungsprozessen- Langenbecks Arch. Chit» Suppl» 151, ¢1984)5Thus, during immunologically mediated stimulation ef gastric digestive processes, besides agressive factors like acid also zytoproteetive mechanisms like increase of blood flow and mucus secretion,, are initiated» Based upon these results further experiments were carried out concerning mediators of the immunological stimulation of gastric functions using the model of isolated antrum-muscular perfusion in dogs. Gastrin and prostaglandi ne E„ and determinations were performed in an intravenous effluent.

The supernatant of fluoreslc duct lymph, collected after antigen administration in immunized animals, stimulated gastrin release in isolated antrum perfusion. This activity was missing in the (Lymph of non-isnmunised animals when the same antigen was administrated into the stomach, (Teichmann, R-K-, Pratschke, Ε., Grab, J . , Tutert, J ., Enders, G„, Brendel, M.: GastrInstI mu Iierende Mirkung von Ductus-thoracicus-Lymphe nach gastrointestinaler Immunreaktion. Langenbecks Arch- Chin. Suppl. 271 (1985)).

Thus, it has been shown that soluble mediators are responsible for the antigen induced gastrin release.

The mucosa mast celt products Mistamin and Leukotrien C^, being released during the Immunological reactions, significantly inhibit antral gastrin release. Gastrin Itself stimulates the release of Histamin so that these results, for the first time, show local antral negative feed-back mechanism between the 6-cell and the mast cell CPratschke, E«, Teichmann, R.K., Grab, J., To*ert? E-, Brendel, fej„: Oer EinfluB von JlastzelIprodukten auf die Gastrinfreisetzung. Langenbecks Arch. Chir. Suppl», 295, Cl 985)5.

Simultaneous to the Inhibition of gastrin release by Histamin and Leukotrien C^, there is a release of endogenous prostaglandin® E^ and F^» In the model of isolated antrum perfusion it could be shown that prostaglandin® E^ inhibits the gastrin release. This eight indicate that the inhibition of gastrin,? by mucosa aasi cell products, may be mediated by endogenously released prostaglandines (Pratschke, E«, Teichmann, R.K., Grab, J., Enders, G„, Srendel, M.: Endogenes Prostaglandin £2 als Regulator der Gastrinfreisettung. Ins KongreBbericht der 26. Jahrestagung der osterr.

Gesellschaft fur Chirurgie. Hrsg-s F. Helmer, E. Horcher, Styria-Verlag, Gras, 131, 11985)).

During the process of antigen recognition involving antigen presenting cells lymphokimes Interleukin 2 and Gamma i nt erferon are released. Both stimulate gastrin. Thus, for the first time, stimulation of a gastrin by mediators of immuno competent cells was shown (Pratschke, E«, Teichmann, R-Κ., Grab, J., Hammer, C- Brendel., M.: Gastrinfreisetzung durch Rediatoren immunkompetenter Zellen. Langenbecks Arch. Chir. Suppl,., 261 11986)).

Immunofluoresence microscopic studies of the antrum of the perfusion with Interleukin 2 and Gamma interferon, using monoclonal antibodies against class 2-antigens of the FJHC complex, revealed further that, after the perfusion with antigenic structure of DLA Class 2 (Dog-Leukocyte antigens), same are expressed on epithelial cells, macrophages, probably also on intraepithelial T-Lymphocyte. There was no expression of those two antigens after perfusion with Interleukin 2.

Therefore, Interleukin 2, but also Prostaglandines and other substances, may serve as mediators of this immunological reaction.

Thus, the above-mentioned antigen induced reactions are discussed for the development of ulcers and aay also be of significance for th® total gastrointestinal tract.

Whenever antigen induced increase of the antrum blood flow, gastrin and mucus secretion is missing, or disturbed (Local Iy, the resulting acid may not be suf f i ciently buffered and transported back in case of lack of increase of blood flow, resulting in penetration of the acid into the mucosa and possibly inducing local ulcers there.

Investigations on th® immunological stimulation of gastric reactions also showed that after immunological stimulation with a synthetic antigen, besides the antigens specific in view 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 was noted which has a protective effect by inhibition of acid and pepsinogen secretion, (Kramling, H.-J., Teiehmann R.K., Merkle, SU, Enders, 6., Brendel, M.: Gastrale Protektion durch imraunologisch induzierte Somatostatinfreisetzung, 153. Tagung Vereinigung Niederrheiniseh-Westf£1ischer Chirurgen, E This data results in a completely new far reaching, and so far not recognised, possibility to specifically influence digestiv© processing as a protective mechanism concerning all disorders and diseases within the gastrointestinal tract- In human medicine, diseases like erosions,peptic ulcers and, last but not least, cancer are -of greatest interests. Ira veterinary medicine interest is mainly in the critical transition phase of young animals following weaning and subsequent contact with high energy food because of occurrence of erosions and peptic ulcers during this -phase» There is a protective induction of functions..of. the. ^aosa^tss^Hsksaos^^sfi^ryeg'giShs'a^Ymmafiasaa^t functions and protective physiologic digestive processes, which may be used in s broadest .sense; this is the use ot an antigenic substance for the prophylaxis and therapy of disorders and diseases of the gastrointestinal tract of animals and humans inducing protective physiological digestive processes -after an oral challenge of the animal or human with the antigenic substance following preceding i rnmuni sat ion.

When applying an antigenic substance, as outlined by the invention, the stimulated gastric functions may work as follows: After immunisation of the stomach with an antigen which is present in the food the subsequent contact of this antigen, in the food, with the gastric mucosa, the antigen will be recognized by the so-called antigen presenting cells, for instance, macrophages and presented to the T-lymphocytes by the help of class 2 antigens- An indication for a lympocyte mediator immunological stimulation of the above-mentioned gastric functions was demonstrated in an experiment in which immunized dogs were irradiated by an 80 Rad of an ultra-hard X-ray resulting in an inhibition of the Immunologically stimulation of gastrin release based upon destruction of lymphocytes- The capability of the gastrin cell to be stimulated, however, is not effected by the irradiation.

Following the contact of the antigen with the stomach, being immunized as outlined above, there is a type of cascade of release of Interleukin 1 and stimulation of T-Helper-Lymphocytes- The Helper sells themselves will release Interleukin 2 and Gamma interferon, two lymophokines which were shown to be able to release gastrin® A significant protective physiological mechanism, namely the increase in mucosa blood flow representing one of several steps of the cascade of protective digestive processes, may be caused by mediators of asst cells, especially Histamine. Besides the expression of Ίθ class-2-amtigens on antigen presenting cells of the antrum, Gamma interferon also induced the expression of these antigens on epithet cells. Possibly this additional recruitment of epithet cells 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 shown in Figure 1.

The ulcer protective effect of antigen specific stimulation of digestion has been demonstrated in an animal model® ’Using the model of the 1SHAY""Ulcer (Shay® H., Komarov, S.A®, Pels, S.S., Mekanze, 9®, Gruenstein, H., Siplet, H«: A simple method for the uniform production of gastric ulceration in th© rat. Gastroenterology 5, 43, (1945)), the influence of antigen stimulated regulation of the digestion was investigated. Hale Mister-Rats were immunized systemically with a Hapten. Afterwards, the Hapten was administered into the stomach and ®s control sodium chloride was given® In further control studies, the Hapten or sodium chloride, respectively was also administered in non-iraraunized animals. These rats being treated in such a way were investigated «acroscopically and microscopically to check gastric lesions after their death but not later than 18 hours® There was a significantly less expression or number of perforations and large wall necroses in the core ventriculous in the group of animals which had been immunised with the Hapten (Figure 3,5.

Thus, using the model of the SHAY-Ulcer the protective effect of an antigeo specific stimulation of the digestion could be demonstrated. Although there is ®n ulcerogenic effect by release of gastrin and, consecutively of acid, as an aggressive factor, simultaneously there is also stimulation of protective mechanisms, for instance, an increase in blood flow, mucus secretion, possibly through release of Somatostatin, Prostaglandines and other mediators which finally prevent the occurence of severe secretions of the gastric mucosa or gastric wall. This ulcer protective effect of an antigen induced regulation of the digestion, as shown here, may teleologically well be regarded as a "logical" regulation within physiological 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 peptic ulcer may well be understood as a local failure of the stomach to be ready for antigenic reactions beside other known causes. The stimulation of gastric immunological mechanisms by immunization with antigens, therefore, results in a completely new possibility for the prophylaxis and therapy of disorders and diseases within the gastrin intestinal tract of animals and humans offering broadest indications. The essential, other advantage of use of antigenic substances, for this purpose, is based on the fact that the antigens being used are biological substances-with which the aamaal as well as the human being comes into contact naturally and, thus, there is no aggressive effect en physcologie®l processes but, moreover, an induction of a logical protective physiological process in a natural Manner and, thus, same is without side effects1 1 In respect of the mentioned mechanisms of the immunologieat stimulation of the digestion, it is, of course, of special advantage to use such substances as antigens which later will also be included within food. foilowing immunization of the animal or the human with the antigenic substance a level of factors, within protective effect, will be increased in such a way that disorders or diseases of the gastrointestinal tract will be prevented or treated. Thus, plant proteins, preferrably from maize or soya bean, but also animal proteins, are especially suitable for the immunization 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, which were additionally administered whey protein via the drinking water during the entire fattening period until slaughtering of the pigs after a fattening of 10 weeks, entailed that in particular serious changes of the mucous membrane in the animals treated, as described above, occurred less frequently by 7ΌΖ than in a control group. The whey protein should preferably be present in a 3 to 9% whey protein solution and especially preferably in a 6Z whey protein solution.

Human proteins, which are components of blood or which may be found in serum or other blood derivates, for instance, hemoglobin are suitable to be used as antigenic substances in th® sens© of th® invention.

Also other antigens, which do not necessarily correspond with substances being present in the ingested food, may be suitable for the induction of the immunological situation which is responsible for the above-mentioned induction of mechanisms. for instance, in ease of Immunization with proteins derived from the envelopes of virus, an Immunization may be possible, which will work In the sense of the above-described stimulation of the digestion, but also way cause an Immunization against virus.

Same Is true for antigenic substances which are present in microorganisms with which the gastrointestinal tract may come into contact in one way 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 always 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 contains antigenic determinants for the Induction of an immune response, is especially preferred because then a special and directly applicable prophylaxis and therapy will be possible.

A well suitable substance for the induction of the Immune 2b situation to induce the protective mechanisms are Haptens, preferably nitrophenyl acetic acid (NIP). This substance has shown its special usefulness in the experiment to Induce the Immunization, which prevents the development of mucosal lesions after intraluminal challenge of an antigen specific stimulation of the stomach. Especially with this substance, an immunologically mediated ulcer protection was shown for the first time.

Preferably, the above-mentioned nitrophenyl acetic acid is used by coupling same to a carrier protein, namely ovalbumin.

As outlined in the description above, immune modulatory substances are in the equilibrium state between the aggressive and protective digestive mechanisms. The addition of immune modulatory substances together with 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 pharmaeeutical conf sctioning 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 application. Concerning the enteral confectioning, 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 intramuscular and/or sub-cutanous applications.

In the veterinary medicine, a preferred use of the antigenic substances is to immunize piglets with the antigenic substance about two weeks after birth. As has already been mentioned above, piglets suffer from peptic ulcers after the weaning and the change to semi-liquids energy-food. They may even die from these ulcers., Treating these piglets in this critical phase with antigens which later will be present in the food prevents, or at least, reduces damages- The proteins being used as antigenic substances are biological substances which, after application, will be metabolized completely without residue or being excreted in only small amounts.

An especially preferred treatment of the piglets is to immunize them intramuscularly, preferably the first injection being about two weeks after birtli; a second injection about four to five weeks after birth and a third injection shortly before changing of food- Xn respect of their practicability, the animals could then be immunized when they receive iron-injections during breeding., The .antigenic substances may be packed in a kit which already contains ironed hen the piglets receive the food which contains said proteins, with which the animals were immunized, the antigen may also be given to the drinking water in order to further induce and maintain the protection of the gastric mucosa by the described immunological mechanismsXn human medicine, the us® of antigenic substances is preferably suitable to prevent or reduce erosions, peptic ulcers or cancer in the human gastrointestinal tract. The increased production of mucos may prevent the mucosa of oncogenic substances- furthermore, oncogenic substances, which are known for the organism, may become inaffected by the induction of the digestion.

Also, there is a new preferred and broad spectrum of indications in the prophylaxis concerning diarrhoea in travellers who come into contact with food products during their trip which are, so far, unknown by their gastrointestinal tract, this means that the gastrointestinal tract, so far, had not been in contact with such food products. The lack of induction of digestion may cause passage of protein into deeper parts of the gut with subsequent possible overgrowth of b®ct®ri a» Xt is also a well known problem in the breeding of domestic animals that following transportation of the domestic animals to places at which th® breeding will be continued after the first week of life, the changes in the environment and food may cause significant diarrhoea which, again, may be the cause of other bacterial or viral diseases of the whole gastrointenstinal tract.

Immunization of these animals with antigenic substances, being known to be present later in the food, may prevent these disorders and diseases.

One possible use of an antigenic substance is to sensitise piglets parenterally as of the third week at intervals of fourteen days with 2 ml each of the 62 whey protein solution without the addition of adjuvant that is to say on the whole with 4 snI. After weaning of the piglets in the second week and gradual change over from pig breeding feed to coarse grain fattening fodder the piglets were brought into another pig sty and kept, in groups in loose housing boxes, from this time on approximately as of the tenth week, all pigs were exclusively fed with very finely ground fattening feed. In literature the administration of such feed is indicated, besides stress factors, as the decisive cause of the formation of mucosal changes, such as hyperkeratoses, erosions or ulcers in the gastric mucous membrane of the pig30 The pigs were administered 3 to 62 whey protein with the drinking water during the entire fattening period until the slaughtering of the pigs, after a fattening of ten weeks16 In animals which have been treated in this way changes of the mucosa-like erosions appeared much less frequently.

In the same way as the antigenic substances have.been used for .the treatment of piglets, calves saay be immunized parenterally and/or enterslly in their critical phases of is preferred to give intramuscular injections oi the antigen in calves within about the first four weeks after birth, a second injection at about the sixth week and a third injection in about the eighth to tenth week when changing to calf food.

Within the first weeks of breeding, the calves receive milk and calf food and, generally, energy food from the eighth to tenth week. From the time of changing to the high-energy food, the antigenic substance is added, preferably to the food and/or drinking water to use and/or maintain the level for the protection of the intestinal tract by the described immunological mechanisms.

The immunization is preferably performed also with adult ruminents. Bulls and cows increasingly suffer from gastric ulcers of different stages during the high lactation period. These ulcers are also especially considered as th® cause of subsequent infectious diseases of the whole gastrointestinal tract in adult cattle.., Enteral or parenteral treatment of animals wi th the antigenie substances may prevent these damages.

Another field of using the antigenic substances is the treatment of domestic poultry, for instance, chicken, turkeys, geese, ducks and turkey-cocks- The major sources of food, for the breeding of domestic poultry, ar© grain granules, fish flour, soy® and grinded peanuts» To induce the unisation, In respect of the above-mentioned food substances, a parenteral injection of the antigenic substance will be performed on the first day after hatching because of pracical reasons. Later, imm uninations for maintenance of a preceding immunization may be performed by application of the antigen.via the dri nki ng water within the early days or weeks of life.

The current invention will now be described, in more detail, in the following Figures and Examples.

It is shown in E.7.SJ5£JL_2, ’ The regulation of antigen stimulated gastrin release.

Luminal antigen is recognized by antigen presenting cells and may stimulate mast cells as well as T-lymphocytes among others. The T-cell products Gamma Interferon and Interleukin 2 cause release of gastrin which, in turn, stimulates Histamin. The mast cell products Histamin, Leukotrien and Prostaglandine Eg again inhibit the gastrin release (from negative feedback). The vagus nerve may also influence the T-cell systems and the antigen presenting cells. £xs jure^Z: Ulcer index using the model of the alcohol ulcer of the Γ Rats being immunized with NIP show a significantly lower ulcer Index following intragastric application of the antigen.

Moure. 5» Percentage of animals with lesions, induced by the SHAY-Ulcer model in controls and in animals immunized with NXP-ovsIburnin.

The intragastric application of ΝΙΡ-human Gamma-globin (NXP-HGG) results In a significantly lower expression of peptic ulcers in Immunized animals.

Figure. ,4; Quantitative changes of the evaluation scales (0 and 4-+O after a pathological-anatomic evaluation of the gastric mucous membrane of pigs in the area of pars proventricularis after a 10 week feeding of very finely ground feed rations (Z). exaragile.j!.

The purpose of the described examples was to investigate the effect of the immunological processes on the other induction concerning stimulation or protection.. fiale Hi star-Rats <250-3S0gS wear® Immunized systemicalty with KIP-OA (Nitrophenyl «acetic acid-Ovalbumin). To induce ulcers, 1 ml of absolute alcohol was administered into the stomach under ether anesthesia. Fifteen minutes prior to the alcohol application, one group (I) of the animals <«=125, -received the antigen KIP (coupled to HGG as carrier-protein), Group II Cns125, the carrier protein HGG alone. Non-immuni zed animals, six having received NIP-HGG (III), a further six HGG (IV), proved as further controls. An hour after the adraini strati on of alcohol, the lesions were determined after the animal was killed and the stomach removed. The lesions were measured macroscopically by estimatation of the length of the hemorrhagic lesions.

Animals having'been immunised systemically with NIP developed significantly less mucosal lesions after oral NlP-application and subsequent administration of alcohol (p " 0,02) than all control groups.

Table 1 Immunisati on Intragastric Length of Administration of hemorrhagic Lesions I (n~12) NIP NIP 24 11 (n=125 NIP HGG 57 III (n=6) non—imm. NIP 72 IV (n=65 non-imm. mn/* fi» into fa 78 The intraluminal antigen specific stimulation. of the stomach in immunised Wistar-Rats significantly reduced the development of mucosal lesions. Therefore, for the first time, an immuoologically mediated ulcer protection is' shown (Figure 25., g&asalejs The immunization of male Wistas—Rats (250-350g) was performed «sing MIP-OA (MitrophenyI acetic acid-Ovalbunin). Pylorus ligated rats'(SHAV-Ulcer) served as ulcer model. following ligation of the pylorus under chloralhydrate anesthesia, the immunized rats received the antigen NIP (coupled to human gamma globulin) directly into tube stomach (test group). The control animals received only the carrier protein HGG. Another control group consisted of sham operated animals (without pylorus ligation). Eighteen hours after the operation, the changes in the stomach wall were investigated macroscopically and microseopically. The lesions were defined as follows: Perforations, confluent transmural ulcerus and all necroses of more than 0,5 cm in diameter20 Animals which were systemically immunized with NIP 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.

Tabie_2 NIP-specifically immunised animals Int ragast ri c Application of Proportion of Animals showing Lesions Test group (ns243 Control group NIP-HGG 7/24 HGG 14/23 NIP-HGG resp.HGG Ο/ό resp. 0/6 The antigen specific stimulation of gastric functions result in a significant reduction of wall lesions in a defined ulcer model (Pylorus ligation) (Figure 3.,)., lE%ggpJ-e.,5 Procedure.of Immuirn Ziati on of .Pickets Supposedly the antibody production in piglets starts from the second week and regarding the practicability, the piglets are immunized and receive iron injections during their breeding by th® breeder™ A biologic protein or s synthetic antigenic immune stimulating substance will be used as antigen» It will be metabolized without residues following digestion by the piglets or it will be excreted, as such, in only small amounts.

For the present immunization of the piglets whey proteins, namely as a 62 whey protein solution, were administered to the piglets without the addition of an adjuvant or during the later course of immunization as a 3 to 62 addition to the drinking water.

Thirty pigs were taken as a test group and thirty pigs as control animals for an examination of the prophylaetic effect of the whey proteins administered as antigens regarding gastral mucosal changes- During the test five pigs of the test group and two of the control animals died as piglets or during change-over.

The test animals were pretreated according to the following scheme. Piglets were sensitized parenterally as of the third week at intervals of 14 days with 2 ml each of a 62 whey protein solution without the addition of adjuvant that is to say on the whole with 4 ml.

After weaning of the piglets in the second weej^ and gradual change over from pig breeding feed to coarse grain fattening fodder both the test and the control animals were brought into another pig stay and kept in groups in loose housing boxes» From this time on, approximately as of the 10th week, all pigs were exclusively fed with very finely ground fattening feed. In literature the administration of such a feed is indicated besides 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.

Both groups of animals were administered 3 to 6% whey protein with the drinking water during the entire 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 was evaluated according to patholog!cal-anatorn!c criteria.

Table 3 shows the frequency of the evaluation scales ¢0 to ***) after a 10 weeks feeding of very finely ground feed rations and their percentages i%). 15 Table 3 0 4- 4--5- 4-4-4- 20 6 21,4% ό 21,4% 8 8 28,6% 28,6% control group n = 28 10 7 6 2 25 40% 28% 24% 8% test group n ~ 25» Xn Figure 4 it is graphically represented that In the animals treated with whey protein twice as much animals had an Intact mucous membrane than in the controls (the two left columns In Figure 4 with the designation "'O'* which is used for an intact mucous membrane). The two right columns of Figure 4 reveal on the other hand that serious changes of the mucous membrane, e.g. erosions, which are designated with "4-4-4--’ occurred by 70% less frequently than In the control.

The exact figures of the macroscopic evaluation of the gastric mucous membranes of the experimental animals as they are revealed by table 3 show that low grade, flat hyperkeratoses (designated with M+") or moderate hyperkeratoses with pointed serrations (designated with occurred with the same frequency in both groups.

However, the changes with ’+4·i.e. the erosions, are decisive, whose prevention in this test with pigs was possible still more clearly than in the experiments described in examples 1 and 2.

Exaaole 4 Performance of Immunisation of Cattle As antigen, a protein is used which is 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 in the 'high-energy food which is given to the calves in about the eighth to tenth week after birth. The ulcerative lesions preferably develop during the change to high-energy food, usually at an age of four to ten weeks., These disorders are in connection with the mentioned lack of immunization of animats regarding the proteins being present in the high-energy food. The main constituents of the high-energy food for ruminents are mainly corn, wheat, soya, barley, grassilage and hay» These constituents may change according to the degree of intensive breeding.

The first intramuscular injection of the used antigenic substance wilt be given in the first four weeks after the birth of the calf. A second injection will follow in the sixth week and a third injection at the time when high-energy food will be given in the eighth to tenth week» Ση the first week of breeding the calves receive milk and starter diet. They will receive high-energy food about the eighth to tenth week» Then, at the time of changing to the high-energy food, the antigenic substance wilt be added to the food and/or drinking water to use or establish the immunologi cal protection of the gastrointestinal tract by the above-described immunological mechanism.

The dose of the antigenic substance should be about 0,1g test substance per kg bodyweight 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 in a dose of up to 1 g per 100 ml water.

Performing this procedure, the ulcerative lesions in the gastrointestinal tract of the calves will be prevented or reduced» According 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 within the first weeks after birth. The frequently-occurring disorders or diseases of the gastrointestinal tract, during these environmental changes, especially the diarrhoea, may well be prevented by the new mechanism- Diarrhoea can be the cause of other bacterial or viral diseases in the gastrointestinal tract.

Concerning the use of antigenic substances in adult ruminants, the required antigenic substances will be applied in bulls and cows during the period of intensive feeding and high lactation. Thus, also in adult animals 2ό the more common gastric ulcers, of various stages, may be prevented or reduced.

Performance of immunization of poultry Similar to the immunization of pigs and cattle, domestic poultry, for instance, chickens, turkeys, geese, ducks or turkey-cocks, will be immunized with antigenic substances in the above-mentioned way. The antigenic substances will consist of grain, fish flour, soya or peanuts according to the major sources of food during the later breeding.

The parenteral injection of the antigenic substance is performed on the first day after hatching due to practical reasons. This first immunization will be intensified established by later application of the antigenic substance by putting the antigenic substance into the drinking water in an ent raI form about one week after the hatching.

Claims (18)

1. Use of at least one antigenic substance for the preparation of a medicament for the prophylaxis and treatment of disorders and diseases in the digestive tract of animals and humans, in which method after prior immunization or sensitization by the antigenic substance(s), a subsequent gastrointestinal administration of the antigenic substance(s) in a suitable formulation is carried out, with the following exceptions: (a) a method for preventing diarrhoea in poultry, as a consequence of a coccidial infection by rearing them with a feed which contains added, live, sporulated oocysts of at least one coccidial species to which the poultry is susceptible, the oocysts being present in a concentration which is sufficient only to induce a subclinical infection in the poultry; (b) a method for the prophylaxis of intestinal diseases in pigs by oral immunization and additional oral administration of heat-inactivated antigen formulations of E. coli strains, which lead to intestinal diseases; and (c) a method for the prophylaxis and treatment of intestinal diseases in pre-ruminant calves by oral immunization and additional oral administration of heat-inactivated antigen formulations of E. coli and Salmonella strains, which lead to intestinal diseases.

2. Use of at least one antigenic substance according to Claim 1, in which the antigenic substance is vegetable protein, preferably from maize or soya.

3. Use of at least one antigenic substance according to Claim 1, in which the antigenic substance is animal protein.

4. Use of at least one antigenic substance according to Claim 3, in which the animal protein is whey protein.

5. Use of at least one antigenic substance according to Claim 4, in which the whey protein is used as a 3-9% strength, preferably 5% strength, whey protein solution. - 28 5. Use of st least one antigenic substance according to Claim 1, in which the antigenic substance is human protein, preferably from blood or blood constituents, namely serum, plasma or haemoglobin.

6. 7. Use of at least one antigenic substance according to Claim 1, in which the antigenic substance is viral protein.

7. 8. Use of at least one antigenic substance according to Claim 1, in which the antigenic substance is a microorganism or a constituent of a microorganism.

8. 9. Use of at least one antigenic substance according to Claim 8, in which the antigenic substance is a bacterium or a component of the cell wall of a bacterium.

9. 10. Use of at least one antigenic substance according to Claim 8, in which the microorganism is a representative of the protozoa.

10. 11. Use of at least one antigenic substance according to Claim 1, in which the antigenic substance is a synthetic protein, preferably a short peptide, which contains antigenic determinants for triggering the immune response.

11. 12. Use of at least one antigenic substance according to Claim 1, in which the antigenic substance is a hapten, preferably nitrophenylacetic acid (NIP).

12. 13. Use of at least one antigenic substance according to Claim 12, in which the hapten is coupled to carrier proteins.

13. 14. Use of at least one antigenic substance according to one of Claims 1 to 13, in which immune modulators are used in addition to the antigenic substance.

14. 15. Use of at least one antigenic substance according to one of the preceding claims, in which the antigenic substance is produced for administration by the intestinal route, preferably as an aerosol, if appropriate with the addition of pharmaceutically tolerated auxiliaries. - 29

15. 16 Use of at least one antigenic substance according to one of Claims 1 to 14, in which the antigenic substance is produced for parenteral administration, preferably intramuscular and/or subcutaneous administration, if appropriate with the addition of pharmaceutically tolerated auxiliaries.

16. 17. Use of at least one antigenic substance according to Claim 15 or 16, for the prophylaxis or treatment of erosions, ulcers or carcinomas in the human digestive tract.

17. 18. Use of at least one antigenic substance according to Claim 15 or 16, for the prophylaxis of diarrhoea caused by foodstuffs or feedstuffs with which the human or animal digestive tract has not yet come into contact.

18. 20. Use of an antigenic substance as claimed in Claim 1 substantially as described herein with reference to the Examples. TOMKINS & CO. ccboy PROF. DR. REINHARD K. TEICHMANN, and PROF. DR. WALTER BRENDEL, and PROF. DR. HANS-GEORG LIEBICH.