Partial Cell Functions

Nobel Lecture, II December 1908

Partial Cell Functions

PAUL EHRLICH

The history of our knowledge of the phenomena of life and of the organized world can be divided intotwo main periods. For a long time anatomy, and particularly the anatomy of the human body, was thealpha and omega of scientific knowledge. Further progress only became possible with the discovery ofthe microscope. A long time had yet to pass until, through Schwann, the cell was established as the finalbiological unit. I shall not describe here the immeasurable progress made by biology in all its branchesowing to the introduction of this concept of the cell. For this concept is the axis around which the wholeof the modern science of life revolves.

It is, I think, a generally acknowledged and undisputed fact that everything which happens in thebody, all assimilation and disassimilation, must ultimately be attributed to the cell alone; andfurthermore, that the cells of different organs are differentiated from each other in a specific way and onlyperform their different functions by virtue of this differentiation.

The results presented here are mainly based on histological examinations of dead and living tissues,with, of course, valuable contributions from the neighbouring sciences—physiology, toxicology, andparticularly comparative anatomy and biology. Yet I am inclined to think that the limit of what themicroscope could and has done for us is now approaching and that for a further penetration into theimportant, a\\-governing problem of cell life even the most highly refined optical aids will be of no use tous. The time has now come to penetrate into the most subtle chemistry of cell life and to break down theconcept of the cell as a unit into that of the cell as a great number of individual specific partial functions.But since what happens in the cell is chiefly of a chemical nature, and since the configuration of chemicalstructures lies beyond the limits of the eye's perception, we shall have to find other methods ofinvestigation for this. This approach is not only of great importance for a real understanding of the lifeprocesses, but also the basis for a truly rational use of medicinal substances.

The first advance in this complicated field came about indirectly, as so often happens. After Behring'srenowned discovery of the antitoxins I had set myself the task of penetrating further into the mysteriousnature of this process, and after long labours I have succeeded in finding the key to it.

As you know, the function of stimulating the formation of antibodies belongs to one particular groupof poisonous substances only, to the so-called toxins. These are metabolic products of animal and plantcells: diphtherial toxin, tetanus toxin, the phytotoxin of jequirity, ricin, snake venom, and so on. None ofthese substances can be made to crystallize, and they obviously belong to the class of protein substances.The toxin is generally characterized by two properties: (i) its degree of toxicity; and (ii) its ability tostimulate the production of the specific antitoxin in the animal body.

My quantitative investigations of this process have shown that the toxins, especially the solutions ofdiphtherial toxin, will—either spontaneously if left standing for some time, or through the action ofthermal influences or certain chemicals (iodine)—change in such a way that they are more or lessdeprived of their toxicity but retain their ability to produce antibodies. Furthermore, it has becomeobvious that the products of this transformation, which I call toxoids, and which my eminent friendProfessor Arrhenius has also encountered in his numerous experiments, have still retained the ability toneutralize the antitoxin in a specific way. Indeed, in favourable cases I and others have succeeded inproving that the transformation of toxin into toxoid can be a perfectly quantitative one, so that a giventoxic solution will combine with exactly the same amount of antitoxin before and after thetransformation into toxoid.

These facts permit only one explanation, namely that there must be two differently functioning groupspresent in the toxin. One of these, which is also preserved in the 'toxoid' and must therefore be consideredthe more stable, must have the ability on the one hand to stimulate the formation of antibodies in theanimal body by immunization, and on the other to neutralize antibodies in the test tube and in vivo. The

The Nobel Lectures 5

relations between toxin and its antitoxin are strictly specific—tetanus antitoxin neutralizes only tetanustoxin, diphtheria serum only diphtherial toxin, snake serum only snake venom, to mention just a fewexamples out of hundreds. For this reason it must be assumed that these antitheses enter into a chemicalbond which, in view ofthe strict specificity, is most easily explained by the existence of two groups withseparate configurations—groups which according to Emil Fischer fit each other 'like a key in a lock'.Considering the stability ofthe bond on the one hand and the fact on the other that neutralization occurseven in very great dilutions and without the help of chemical agents, it must be assumed that this processis to be attributed to a close chemical relationship and probably represents an analogue to actualchemical syntheses.

More recent investigations have in fact shown that chemical action can break down the product oftheunion, the neutral combination of toxin-antitoxin, into its original components. Morgenroth, forexample, has shown, that for a number of toxins—such as snake venom and diphtherial toxin—thecompound can be separated again into its original constituents, by the action of hydrochloric acid. Thisis the same mechanism as that by which in pure chemistry stable compounds, such as the glycosides, canbe broken down into their two components: sugar and the basal aromatic complex, by the action ofacids. These investigations have shown that the stable group of the toxin molecule, which I callhaptophore, can exercise a strong specific chemical action, and thus the obvious assumption is that itmust be precisely this group which causes the adhesion ofthe toxin to the cell. When we see how somebacterial poisons produce disturbances only after weeks of incubation and then damage the heart orkidney or nerves, when we see how animals suffering from tetanus present contractions and spasms formonths, we are forced to conclude that all these phenomena can only be caused by the adhesion of thetoxic substance to quite definite cell complexes.

I therefore assumed that the tetanus toxin, for example, must unite with certain chemical groupings inthe protoplasm of cells, particularly the motor ganglion cells, and that this chemical union is a necessaryand sufficient cause ofthe disease. I have therefore simply called such cell groupings/7OMO« receptors orsimply receptors. Wassermann has been able to prove my view correct in every detail in his notedexperiments which produced the first evidence that the normal brain was able to render innocuous givenquantities of tetanus toxin that were introduced. Many objections have been made to these experiments,but none has proved valid, and I believe that I may state definitely that certain specific groupings must infact exist in the cells which fix the poison. That these, the cell's receptors, which produce the fixation,react to the haptophore part ofthe toxin can be deduced from the immunizations through toxoids, wherethe haptophore group is the only one which has been preserved. But since this haptophore grouping ofthe toxin must have a highly complex and individual stereochemical structure, and since it reactssimultaneously and in the same way to the cell receptors and the antitoxin, it must be concluded from thisthat the group in the protoplasm, the cell receptor, must be identical with the antitoxin which is containedin the serum of immunized animals, for a really well-made key will not open different locks at the sametime. As the cell receptor is obviously pre-formed, and the artificially produced antitoxin is only theconsequence, i.e. secondary, one must assume that the antitoxin only consists of discharged componentsof the cell, namely receptors discharged in excess. The explanation for this is a very obvious one. Thevarious specific cell receptors which take up the snake venom, the diphtherial toxin, the tetanus toxin, thebotulin poison, etc., are not designed to serve as toxic receptors for substances the animal normally doesnot come into contact with. They exist to combine chemically with normal products of metabolism, i.e.to assimilate them. As these receptors, which may be regarded as side chains ofthe protoplasm, capableof assimilation, become occupied by the toxin, the respective normal function of this group is eliminated.Now the fundamental law of tissue defect and its compensation, discovered by Karl Weigert, comes intoplay—the deficiency is not merely compensated, but made up to excess, i.e. there is hyper-regeneration.Finally, if the injections are increased and repeated, so many such groupings are formed in the body ofthe cells that they inhibit as it were the normal functions and the cell disposes ofthe disturbing excess bydischarging it into the blood.

The enormous difference between the amount of poison injected and the antitoxin produced isprobably the most characteristic feature of this process. This is best illuminated by Knorr's statementthat one part of toxin produces an amount of antitoxin capable of neutralizing millions of times theamount of the poison which started the process.

6 The Nobel Lectures

However, there are many who consider the process to be much simpler than this. Straub is of theopinion that it is on the whole analogous to the simpler processes of vital detoxification, for example tothe forming of a sulphuric acid ester from injected phenol, and that the two processes only differ in thatthe phenol-sulphuric acid remains stable in the organism, while the toxin-antitoxin complex in theorganism is not held but is partially destroyed. However, only one component, the injected toxin, is saidto perish, while the other, the product of the reaction of the organism—as something which hasdeveloped in the body and thus is not foreign to it—escapes elimination and remains preserved in theblood and body fluids. By systematic repetition ofthe poisoning it would then be possible to accumulateprotective power in the blood, so that when it is introduced into other organisms it can also protect thesefrom toxic diseases and would thus be acting as a curative serum.

Faced with such a simple explanations, it can only be surprising that this problem has occupied thegreat army of researchers studying immunity for so many years. However, in fact Straub has completelymissed the vital clue, namely that according to his theory a certain amount of toxin would produce onlythe exact equivalent ofthe amount of antitoxin. Fortunately this does not happen in immunization. Onthe contrary, it has been proved much more conclusively—and I refer to my statement about Knorrabove—that one part of poison can produce so much antibody that a millionfold multiple of theequivalent is achieved. This should prove Straub's view to be untenable.

The evidence of this hyper-regeneration indicates the pre-formation and chemical individuality ofthetoxin receptors concerned. Substances which can be constantly formed anew in the cell and mixed withthe blood like a secretion must have a chemical 'individuality'. This realization was the first step whichled to the differentiation of the concept of the cell into that of a great number of separate, individualfunctions. I had assumed from the beginning that the toxin represents nothing more than a nutritivesubstance capable of assimilation, to which is attached—by some sort of accident—a lateral grouping,usually of an unstable nature, which causes the toxic action as such.

This view, which I have held from the beginning, has since been confirmed many times over. Thecomplete independence of the haptophore and toxophore groups has been proved by the discovery ofsubstances which had the ability to produce antibodies, and which were thus antigens, without at thesame time having a toxic effect. I would like to remind you ofthe precipitins, which were first discoveredby Kraus, Tschistowitsch, and Bordet. The important discovery was made that even the genuine proteinsubstances of the animal and plant organism are able, irrespective of whether or not they have a toxiceffect, to produce antibodies with a specific chemical reaction. From this it could be proved that actualnutritive substances also had an antigenic nature, just as my observation had predicted. However, evenamong the poisons produced by nature some have been found which will readily demonstrate theindependence ofthe haptophore and toxophore apparatus. These are the cytotoxins which are normallyfound in the blood serum of higher animals or which can be produced arbitrarily through immunizationwith any type of cell. They differ from all other poisons known to us in their extraordinary specificity,their monotropic action, which is so far only a property of those poisons which are fabricated in thehving animal body. Because of the complexity of their constitution the haptophoric and toxophoricprinciples are obviously different. Thus, the distributive component, the amboceptor, has the function ofconcentrating the active substances on the alTected substrate, through the increase in avidity whichfollows localization. The fact that the animal cells are antigens, although they have no toxic action,proves not only that immunization can be carried out with protein substances in solution, but also thatthe haptophore group is solely responsible for the formation of antibodies.

This discovery and analysis of the specific relations between haptophore antibody groups andreceptors has become ofthe highest theoretical and practical significance in recent serum diagnosis. HereI shall mention only the determination of the agglutination titre which has found its most importantapplication in Widal's typhoid reaction; the differentiation of proteins established by Wassermann andUhlenhuth, which is so important for forensic blood tests; the measurement of the opsonic indexinaugurated by Wright; and the manifold uses which have been found for the process of complementfixation—the scientific foundation of which likewise rests on the principle of the adhesion of theantibody to the haptophore group.

I shall not explore this subject any further now. I shall only conclude that there is a series of nutritivesubstances, probably mostly of protein nature, which find specific receptors in the cells. It is thus


possible, through immunization, to entice these substances into the blood in great abundance in theirmost common forms, the agglutinins, the precipitins, the amboceptors, the opsonins on the one hand,and the antitoxins and antiferments on the other. They then accumulate there in such great numbers thatthey can be thoroughly studied, which is quite impossible within the cell formation. The analysis of suchprocesses has led, for example, to the study of the type of link between toxin and antitoxin, and to thediscovery of the very complicated action of the amboceptors.

Of course this does not solve the secret of life itself, which may be compared with the intricatemechanism of a very complex clock. However, the possibility of removing individual wheels from theclockwork mechanism and studying them represents an advance over the old method of breaking thewhole work into pieces and then trying to deduce something from the conglomeration of broken parts.

I shall call all the receptors which are able to and designed to assimilate nutritive substancesnutriceptors. I regard these nutriceptors as the source of the antibodies I have enumerated above, whichare of such great theoretical and practical importance. The complex nature of the organism, the almostunlimited variety and speeifieity of the cell functions, all seem to me to indicate that there exists a wholerange of nutriceptors of different types. From the point of view of immunization these can bedifferentiated into three classes.

1. Those which do not enter the blood in the form of antibodies. This probably applies to nutriceptorswhich serve the very simplest functions, for instance the assimilation of simple fat or sugar substances.

2. Those which enter the blood in the form of the antibodies mentioned and characterized above, thedevelopment of which corresponds to a hyper-regeneration.

3. The third form presents a contrast, since it does not represent a new formation, but a decrease inreceptors. So far, however, we have very little experimental proof of this event. The only known instanceis probably the evidence produced by H. Kossel that after prolonged immunization of rabbits with thehaemotoxie eel serum, the blood corpuscles finally became insensitive to this agent, as though they hadlost the specific receptors.

Now I, in company with my colleagues, Dr Rohl and Miss Gulbransen, have succeeded in penetratingfurther into the nature of the artificial loss of receptors and in illuminating the whole mechanism. Ourwork will shortly be published in a more extensive form; here I would like to emphasize that theexperiments were done on trypanosomes. Franke infected a monkey at my Institute with a certain speciesof trypanosome and then cured it by means of ehemotherapeutic agents, and then again, in order to testthe immunity of the animal, re-infected it with the original strain. Contrary to expectation, the monkeywas found not to be immune, and it sickened again after a very prolonged period of incubation. If micewere treated with blood coming from the infected animal, i.e. containing trypanosomes, they fell ill anddied. However, if the trypanosomes were first removed from the blood of the monkey it became apparentthat the serum thus produced was capable of killing off the original parasites. This revealed that a newstrain of parasites had developed in the monkey which in contrast to the original strain was no longeraffected by the serum—a serum-resistant strain. Similar observations were at the same time recorded byKleine and recently also by Mesnil.

Now if experimental animals which had been infected with a certain species of trypanosome aretreated not with a full sterihzing dose of a suitable substance (arsanil, arsacetin, arsenophenylglycin), butwith a somewhat smaller one, trypanosomes disappear from the blood for a greater or lesser period oftime. The formation of antibodies has occurred in this ease too, as ean be easily proved. The few parasiteswhieh have escaped death now remain in the organs for some time. They gradually adapt themselves tothe anti-substances in the serum, and then, as soon as this has happened, they return to the blood, wherethey increase rapidly and lead to the death of the animal. If the trypanosomes obtained by this methodare transferred to a group of mice which have been previously infected with the original strain, have beencured through the administration of suitable doses and have thus become carriers of the specificantibodies, and to a second group of normal mice, the parasites will inerease in numbers equally rapidlyin both groups. The parasites of the recidivating strain have therefore undergone a biological change inthat they have become serum-resistant.* The change which has thus been produced in the parasites is not

* It is also possible to obtain exactly the same strain in another, much simpler way which consists of infecting themice with the original strain, fully curing them on the second day with a full dose, and then re-infecting them 2-3 dayslater with the same strain. After some time parasites will appear in the blood which fully correspond to those of therecidivating strain.


a superficial one, but may be reproduced unaltered for many months by passage through normalanimals. The recidivating strain retains, unchanged, its property of being resistant to the antibodiesproduced by the original strain and can thus be identified with absolute certainty.

We now wished to obtain an insight into the nature of this process. The explanation for this, which wefound after a number of different experiments, is the following: the original strain contains an abundanceof a certain uniform type of nutriceptor which we shall call group A. When the parasites are killed anddissolved within the organism of the mice the A grouping acts as an antigen and produces an antibodywhich is related to group A. If living parasites are now brought in contact with this antibody, either in thetest tube or in vivo, the trypanosomes will adhere to it. The effect on the parasites is that they undergo invivo the biological change which leads to the development of the recidivating strain. In the new strain theoriginal A grouping disappears and a new grouping, which we shall call B, appears instead. The evidencethat the grouping in the reeidivating strain is new is as follows: if two mice are infected with therecidivating strain—carrier of the B grouping—and then completely healed; if one mouse is then infectedwith the original strain, and the other with the recidivating strain, the re-inoculation with the originalstrain—carrier of A grouping—proceeds smoothly, while re-infection with the recidivating strain willfail at first. This shows that the original and recidivating strain are not identical, or must possess twodifferently functioning groups. We therefore have a typical case of immunization producing a loss ofreceptors while developing a completely new type of receptor.

Whether one calls this change a mutation or a variation is really not important. The main point is thatit can be produced intentionally and artificially and that it is hereditary. But in view of the great interestwhich this particular problem has for biology and the theory of evolution, we have tried to obtain a fullerunderstanding of the process.

First, it was necessary to determine how the trypanosome antibodies influence the parasites. Acommon assumption in immunology would be that these antibodies have a directly toxic action, i.e.contain toxophoric or trypanolytic groups, and that therefore the adhesion as such would necessarilydamage or kill the cell. My colleagues and I have become convinced that this is not the case. In contrast tothe usual species of trypanosomes, whieh contain only one uniform grouping A, B, or C etc. and whichmay therefore be called unios, there is also another type, with two groups in the protoplasm at the sametime (e.g. A and B), which can therefore be called binios. If one such binio A-B is acted upon by theisolated antibody A or B, this has no effect on growth. Growth is only affected if the parasite is occupiedby both anti-substances at the same time. It follows from this that the presence of antibodies does nothave a directly toxic effect on the trypanosomes. This triple experiment also appears to show that theantibody only has an effect in so far as it prevents the intake of nutritive substances through occupationof the group concerned. If in the binio A-B the grouping A is obstructed by the antibody, the parasite cancontinue to multiply through its grouping B. This also proves that the groupings A and B must be chieflyregarded as nutriceptors.

If the amount of antibody is very large the parasite can no longer feed itself at all and dies. A simplemethod of demonstrating this is to mix parasites with varying amounts of antiserum in test tubes. Highconcentrations which stop the intake of food altogether cause the death of the parasites, while at weakerconcentrations, which permit enough life for mutation to be possible, a recidivating strain develops. Thismutation must therefore be entirely due to starvation of the protoplasm, under the influence of whichnew potential structures of the trypanosome develop. Antibodies like those we have just beenconsidering, which have a purely anti-nutritive action, I call atrepsins and I believe they probably play anextraordinarily important role not only for bacteria, but in biology in general.

Most of my colleagues in this field will probably find it easy to accept the idea that there are certainchemical groups in the cell for the reception of the various nutritive substances, once their existence hasbeen definitely proved by the presence of the antibodies. However, a more difficult question is whetherthere are analogous functional groups for the reception of other, less complicated substances as well. Forthe simplest function of the cell, the absorption of oxygen, the problem has in my opinion already beensolved. We know that in the haemoglobin molecule the organically associated iron residue provides theonly loose link between oxygen on the one hand and carbon dioxide and hydrocyanic acid on the other.Thus, we assume certain groupings in the protoplasm of the red blood corpuscles, which form a complexcompound with iron, with characteristic functional properties. The protoplasm of the red blood


corpuscles would thus be characterized by the abundant presenee otferroceptors, which, complementedwith iron, would result in the finished haemoglobin molecule. In a similar way it will also be necessary toassume that the blue respiratory pigment of crayfish contains cuproceptors, and other species probablymanganoceptors. The localization of iodine in certain glandular systems, particularly the thyroid gland,and the evidence that iodine is arranged in certain aromatic lateral chains will also have to be interpretedin this way.

Much more difficult, however, is the question of whether the cell contains such preformedchemoreceptors that react with drugs. This question takes us into the important field of the relationbetween constitution and action, which forms the basis of a rational development of therapy. Only whenwe really know the points where the parasites attack, and have established what I call the therapeuticbiology of the parasites, will it be possible to combat the infectious agents successfully.

I therefore started my studies of the detection of definite chemoreceptors with monocellular livingbeings—protists—because the conditions there are much more favourable to a clear understanding thanthose in the infinitely complicated machinery of the higher organisms. I asked myself the question: do thetrypanosomes possess in their protoplasm definite groups which govern the capture of definite chemicalsubstances?

If a certain substance is able to kill trypanosomes or other parasites in the test tube or in the animalbody, this can only happen because it accumulates in these parasites, but the process itself is notexplained by the establishment of these bare facts. There are very many explanations for it and only whenit is possible to prove that we have here a function open to specific changes and variations, will we haveproof of a preformed formation.

Unfortunately, the method used to provide evidence of the preformation of nutriceptors, namely thetransfer of cast-off receptors into the blood, does not apply for chemoreceptors. This is becausechemoreceptors are much more simply constructed and remain attached to the cell—that is, they are notrejected.

Here it was only possible to advance indirectly, which took us via the drug-resistant strains of thetrypanosomes. Together with my colleagues Franke, Browning, and Rohl, I have shown that it ispossible to obtain by a systematic treatment trypanosome strains which are resistant to the threesubstances which so far are known to be hostile to trypanosomes: compounds of the arsenic series,fuchsin, and the acid azo-dye from the benzopurpurine series, trypan red. These resistant strains have thefollowing characteristics.

1. Stability of the acquired property. This is so great that for instance our arsenic strain, after it hadpassed about 380 times through mice in 2j years, is now even today just as resistant to drugs as theoriginal strain.

2. A principal characteristic of drug resistance is its strict specificity which is distinctive in that itrelates not to one specific compound but to the whole chemical grouping to which this specific compoundbelongs. The strain resistant to fuchsin, for example, is also resistant to a whole series of relatedtriphenylmethane dyes (e.g. malachite green, ethyl green, hexaethyl violet). On the other hand, it hasremained sensitive to both the other types, that is, to trypan red and an arsenical substance. Acorresponding specificity is shown by the strain resistant to trypan red and also that resistant toarsenicals. That there are in fact three different functions here is also shown by the fact that successivetreatment of one and the same strain of trypanosomes with the three above-mentioned substances resultsin a triple-resistant strain, i.e. a strain which is resistant to representatives of all three classes. Such astrain, assuming maximal stability, is extremely valuable for the discovery of new types of trypanoeidalagents. If, for instance, some new substance is obtained which is able to destroy the normaltrypanosomes, it is only necessary to let this substance act upon the triple-resistant strain to find outwhether or not it is a new type of remedial substance. If it is not, the triple-resistant parasites will notdisappear with this treatment, but continue to flourish; but if they do disappear the tested substance doesnot correspond to any of the three types of remedial substance mentioned, and is a representative of anew class of remedial substances. The triple-resistant strain is therefore so to speak the therapeutic sieveby which it is possible to recognize what is homologous, and separate what is different.

A further important question was then to determine how this speeifie drug resistance occurs. Here Iused the atoxyl strain for the experiments. To obtain an exact picture it seemed necessary to investigate


the behaviour of the arsenic resistant parasites in the test tube, removed from all the disturbances andcomplications of the organism. Here a great difficulty soon arose, as the remedial substance used mostoften in therapy, atoxyl (p-aminophenyl arsonic acid), does not have the slightest lethal effect ontrypanosomes in the test tube; even stronger solutions were not sufficient for this. This phenomenon wasall the more striking since according to Koch's investigations the parasites eould be made to vanish in thehuman body within a few hours of injections of 0.5 g atoxyl; a lethal effect had therefore been achievedwith a concentration of 1:120,000.

This was a process which has more recently been termed an 'indirect effect'. It was not difficult for meto find the reason for this phenomenon since I had previously made a thorough study of the reducingpower of the body. We know that arsenic acid in the body is reduced to arsenous acid; we also know thatcacodylic acid is reduced to the foul-smelling cacodyl; it was therefore obvious to start with reduction. Inatoxyl, /7-aminophenyl arsonic acid, the arsenic residue is pentavalent, while in the two productsobtained by its reduction the arsenic residue only has a trivalent action—as in arsenous acid. We thusobtained two different products:

(a) monomolecular p-aminophenyl arsenie oxide

AsO

NH2

and

(b) arising from the reduction of the latter, the yellow diaminoarsenobenzene

As Aso oNH: NH2

In contrast to atoxyl, these substances proved to be highly trypanoeidal both in the test tube and in theanimal body. Even solutions of 1:1,000,000 of the arsenic oxide compound destroyed the trypanosomeswithin 1 h. The closely related p-hydroxyphenyl arsenic oxide has an even stronger effect: 1:10,000,000.

This showed that the pentavalent arsenie residue releases no trypanoeidal function whatsoever, butthat this function is exclusively connected with the trivalent unsaturated state.

More than 60 years ago Bunsen, with prophetic clarity, pointed out that cacodyl, the product ofreduction, is poisonous in comparison with the almost non-poisonous cacodylic acid. From this hededuced the chemical character of the binding of the cacodyl. This agrees well with the fact thatunsaturated carbon monoxide, for instance, and a number of other unsaturated compounds are verymuch more toxic than the corresponding saturated radicals. We shall therefore have to assume that thearsenoceptor of the cells is only able to take up the arsenic residue, which is unsaturated and thereforeadheres easily.

With the aid of such reduced compounds it was now quite easy to examine the atoxyl strain in the testtube. It became apparent that suitable concentrations of the chemicals would still destroy it, i.e. that thiswas not a case of loss of receptors, as we had proved with regard to the recidivating strain. However, acomparison of the lethal dose with that necessary to destroy the normal strain showed that the resistantstrain required a much higher concentration, and that an amount which would destroy the normal strainat once did not, even after 1 h, show the slightest effect on the viability of the resistant parasites.

These test-tube investigations seemed to indicate that the arsenoceptor had been preserved in theatoxyl-resistant trypanosome strain, but that its avidity had decreased, which could be seen from the factthat only through the use of much stronger solutions could the toxic concentration necessary for lethaleffects be achieved; the normal arsenoceptor of the original strain will attract the same amount to itselffrom weaker solutions because of its initially higher avidity.


We have now been able to prove biologically quite clearly that the arsenoceptor does in fact represent acertain function, the avidity of which can be systematically and successively decreased throughimmunization. So far we have been able to trace three different stages in the relationship. Stage I wasachieved by subjecting the parasites systematically to treatment by /7-aminophenyl arsonic aeid and itsacetyl product. We continued increasing the treatment for years, until there was no further increase. Theresistant strain thus obtained was at the same time also resistant to a whole series of other arseniccompounds, like the /7-oxide compound, the urea compound, the benzylidene compound, a number ofacid derivatives, etc.

Because arsenic-resistant strains develop during therapeutic processes, frequently in animals andsometimes in man, and because these naturally completely prevent a successful continuation of therapy,it was now necessary to find substances that could attack the resistant strain and combine with itsreceptors. After a long search we found three compounds altogether, the most important of which wasarsenophenylglycine. With the help of this compound even the arsenic strain I characterized above wascombatted. This can only be explained by the fact that the substance seizes the arsenoceptor like a pair ofpincers. This anchorage, however, opens the possibility of obtaining a still higher resistance to arsenic.We did, in fact, succeed in this, though not without considerable trouble, and derived from the arsenicstrain I at a higher level, arsenic strain II, which was completely resistant to arsenophenylglycine.

Now Plimmer has reeently discovered a preparation, tartar emetic, which at high dilutions alsodestroys trypanosomes. Tartar emetic is the salt of an antimony compound which is closely relatedchemically to arsenic. When we tested tartar emetic on the arsenic strain II we found that the latter wasdestroyed by the tartar emetic. We then treated the arsenic strain II with arsenous acid, so that a thirdstrain developed, arsenic strain III, which had now also become resistant to tartar emetic. I would like toemphasize particularly that this arsenic strain III, which was bred only under the influence of arsenousacid, was resistant to tartar emetic, but not to arsenous acid. This result can only be explained by theassumption that it is arsenous acid which, of all conceivable arsenic compounds, has the strongestrelationship to the arsenoceptor, and that it will probably require the greatest effort or even be entirelyimpossible to produce a strain—and this would be arsenic strain IV—which would be resistant toarsenous acid as well.

To support my view that under the influence and attack of selected compounds there is a successivedecrease in avidity of the same receptor, I could produce many more interesting facts; for example thephenomenon that the trypanosome can also be made resistant directly, with a more effective reagent, i.e.arsenophenylglycine. A strain produced in this way proved, as expected, resistant also to the class ofsubstances with less avidity, i.e. atoxyl, acetyl arsenilate, etc. A panresistant strain would thus beobtained if resistance were created immediately with the most highly effective agents—tartar emetic andarsenous acid. However, our researches seem to show that resistance cannot be produced directly withthese substances; it is only possible to do this indirectly via the previous treatment of strains with phenylarsonic acid derivatives.

The restriction of avidity is of course a chemical process, which allows us to assume that other groupsin the vicinity of the arsenic group concerned develop or disappear, reducing the capacity to react.Perhaps I may give a chemical example. Benzyl cyanide reacts to nitrosodimethylaniline. In order thatthe reaction may take place, heat and a stronger condensation agent, the free alkali, are necessary. If, onthe other hand, a nitro group is introduced into the benzene nucleus, the reacting power of the methylenegroup is heightened considerably; the two substances, nitro-benzyl cyanide and nitrosodimethylaniline,react even in the cold. The introduction of the nitro group has therefore had an accelerating influence onthe reaction. If the nitro compound is reduced to p-aminobenzyl cyanide, it is less capable of reactionthan the original material; the amino group has therefore had a diminishing influence on the reaction,while the acetyl product of the amino compound reacts more or less like the original material.

We can see from this simple example that three different groups, attached to the benzene nucleus in theparaposition, will have no influence whatsoever on the methylene group, or will strengthen or weaken it.The weakening would in our case correspond to the restriction of intensity.

In my opinion the protoplasm can therefore be divided into a large number of individual functionswhich are interspersed among the nutriceptors in the form of different chemoceptors. I think, however.


that these two main groups must be closely interconnected. This becomes apparent from the followingconsideration.

Trypanosomes of different origin, bred in different laboratories, usually show different behaviourright from the beginning towards a certain curative substance. For instance, the trypanosome strain Maide Caderas which I tried first had no resistance to trypan red, and I was therefore able to obtain a curewith this substance. This is still possible even today. Jakimov has had similarly good therapeutic effectsin Russia, while Uhlenhuth observed no influence on his strains. These are therefore innate differences;but that these are not wholly fortuitous is obvious from the fact that even today, after it has passedthrough normal mice for many years, my strain shows the same curability through trypan red as before.In contrast, my Nagana strain could not be cured by trypan red and is still the same today. However,when we made this into a recidivating strain it became apparent that within 14 days this property, whichhad been continued and maintained for years, had altered. This proves that the chemoceptors areconnected with the constitution of the protoplasm and undergo alterations if we alter the constitution ofthe protoplasm by mutation.

The reverse, i.e. whether a change in the cell substance, and particularly its nutriceptors, can beachieved by influencing the chemoceptors, has not yet been definitely established. Browning had indeedobserved and reported that through the serum reaction the fuchsin and atoxyl strains differ from eachother and from the original strain. More detailed investigation, however, has shown further that thesewere not specific changes in connection with fuchsin or arsenic but changes corresponding to therecidivating mutation described above. These changes are due to the fact that during the treatment themice have frequently undergone recidivations which then led to the development of recidivating strains.

I have thus come to the end. I am conscious of the fact that there are gaps in the work I have presented;but how could it be otherwise with a subject that requires such a truly exhaustive study? I wished,however, to show you that we are attacking the problem of how to obtain an insight into the nature ofaction of therapeutic substances. I also hope that if these studies are followed up systematically, it will beeasier to develop a rational drug synthesis. In this respect arsenophenylglycine has so far proved an idealremedy in animal experiments.

For with the help of this substance it is really possible in every animal species and with every kind oftrypanosome infection to achieve a complete cure with one injection, a result which corresponds to whatI call a significant sterilization therapy.

BiographyPaul Ehrlich was born on 14 March 1854 at Strehlen, in Upper Silesia, Germany. He was the son of Ismar Ehrlich andhis wife Rosa Weigert, whose nephew was the great bacteriologist Karl Weigert.

Ehrlich was educated at the gymnasium at Breslau and subsequently at the Universities of Breslau, Strassburg,Freiburg-im-Breisgau, and Leipzig. In 1878 he was awarded his doctorate of medicine for a dissertation on the theoryand practice of staining animal tissues. This work was one result of his great interest in the aniline dyes discovered byW. H. Perkinin 1853.

In 1878 he was appointed assistant to Professor Frerichs at the Berlin Medical Clinic, who gave him every facility tocontinue his work with these dyes and the staining of tissues. Ehrlich showed that all the dye used could be classified asbasic, acid, or neutral, and his work on the staining of granules in blood cells laid the foundations of future work onhaematology and the staining of tissues.

In 1882 Ehrlich published his method of staining the tubercle bacillus discovered by Koch. This method was thebasis of the subsequent modifications introduced by Ziehl and Neelson, which are still used today. The Gram methodof staining bacteria so much used by modem bacteriologists was also derived from it.

In 1882 Ehrlich became Titular Professor and in 1887, as a result of his thesis 'Das SauertstofTbcdurfuis desOrganismus' (The need of the organism for oxygen), he qualified as a Privatdozent (unpaid lecturer or instructor) inthe Faculty of Medicine at the University of Berlin. Later he became an Associate Professor there and Senior HousePhysician to the Charite Hospital in Berlin.

In 1890 Robert Koch, Director of the newly established Institute for Infectious Diseases, appointed Ehrlich as oneof his assistants. It was here that Ehrlich began the immunologieal studies with which his name will always beassociated.


At the end of 1896 an institute for the control of therapeutic sera was established at Steglitz in Berlin and Ehrlichwas appointed its Director. Here he did further important work on immunology, especially on haemolysins. He alsoshowed that the toxin-antitoxin reaction, like chemical reactions, is accelerated by heat and retarded by cold, and thatthe content of antitoxin in antitoxic sera varies so much, for several reasons, that it was necessary to establish astandard by which the antitoxin content could be accurately measured. He accomplished this with von Behring's anti-diphtheria serum and thus made it possible to standardize this serum in units expressing fixed measurements. Themethods used by Ehrlich formed the basis of all future standardization of sera. This work and his otherimmunologieal studies led Ehrlich to formulate his famous side-chain theory of immunity.

In 1897 Ehrlich was appointed Public Health Officer at Frankfurt-am-Main and in 1899, Director of the newlyestablished Royal Institute of Experimental Therapy. He also became Director of the Georg Speyerhaus, which wasnext door to Ehrlich's institute. These appointments marked the beginning of the third phase of Ehrlich's many andvaried researches. He now devoted himself to chemotherapy, basing his work on the idea, which had been implicit inhis doctoral thesis, that the chemical constitution of a drug must be studied in relation to its mode of action and itsaffinity for the eells of the organism against which it is directed. His aim was to find chemical substances which havespecial afi[inities for pathogenic organisms. In the same way as antitoxins make for the toxins to whieh they arespecifically related, these substances would act like 'magic bullets', going straight to the organisms at which they wereaimed.

To achieve this, Ehrlich and his assistants tested hundreds of chemical substances. He also studied the treatment oftrypanosomiasis and other protozoal diseases, and produced trypan red. As his Japanese assistant Shiga showed, thiswas effective against trypanosomes. He also established, with A. Bertheim, the correct structural formula of atoxyl,which was known to have an efficient action against certain experimental trypanosomiases. This discovery revealed away of obtaining numerous new organic compounds with trivalent arsenie, whieh Ehrlich tested.

At this time, the spirochaete that causes syphilis was discovered by Sehaudinn and Hoffmann in Berlin, and Ehrlichdecided to try to find a drug that would be effective specifically against this spirochaete. Among the arsenical drugsalready tested for other purposes there was one, the 606th of the series, which had been set aside in 1907 as beingineffective. However, Ehrlich, learning that Hata had succeeded in infecting rabbits with syphilis, asked Hata, one ofthe researchers at his Institute, to test this discarded drug on syphilitic rabbits. Hata did so and found that it was veryeffective.

When hundreds of experiments had repeatedly proved its efficacy against syphilis, Ehrlich announced it under thename 'Salvarsan'. Subsequently, another arsenical substance, 'Neosalvarsan', was found to be more easilymanufactured and, being more soluble, more easily administered even though it was less efficient therpeutically. Likeso many other discoverers before him, Ehrlich had to battle with much opposition before Salvarsan or Neosalvarsanwas accepted for the treatment of human syphilis, but ultimately practical experience prevailed and Ehrlich becamefamous as one of the main founders of ehemotherapy.

During the later years of his life, Ehrlieh was eoncerned with experimental work on tumours and with his view thatsarcoma may develop from carcinoma. He also worked on his theories of immunity to cancer.

Ehrlich's indefatigable industry throughout his life, his kindness and modesty, his lifelong habit of eating little andsmoking 25 strong cigars a day, a box of which he frequently carried under one arm, his invariable insistence on therepeated proof by many experiments of the results he published, and the veneration and devotion shown to him by allhis assistants have been vividly described by his former secretary, Martha Marquard. Her biography has given us adetailed picture of his life in Frankfurt. In Frankfurt the street in which his institute was situated was named PaulEhrlichstrasse after him, but later, when the persecution of the Jews began, the street was renamed. After the SecondWorld War, however, when his birthplace, Strehlen, came under the jurisdiction of the Polish authorities, theyrenamed it Ehrlichstadt, in honour of its great son.

Ehrlich was an ordinary, foreign, corresponding or honorary member of no less than 81 academies and otherlearned bodies in Austria, Belgium, Brazil, Denmark, Egypt, Finland, France, Germany, Great Britain, Greece,Hungary, Italy, The Netherlands, Norway, Romania, Russia, Serbia, Sweden, Turkey, the U.S.A. and Venezuela, Heheld honorary doctorates of the Universities of Chicago, Gottingen, Oxford, Athens and Breslau, and was also givenorders in Germany, Russia, Japan, Spain, Romania, Serbia, Venezuela, Denmark (Commander Cross of theDanebrog Order), and Norway (Commander Cross of the Royal St Olaf Order).

In 1887 he received the Tiedemann Prize of the Senckenberg Naturforschende Gesellschaft in Frankfurt-am-Main,in 1906 the Prize of Honour at the XVth International Congres of Medicine in Lisbon, in 1911 the Liebig Medal of theGerman Chemical Society, and in 1914 the Cameron Prize of Edinburgh. In 1908 he shared with Metchnikoff thehighest scientific distinction, the Nobel Prize.

The Prussian Government elected him Privy Medical Counsel in 1897, promoted him to a higher rank of thisCounsel in 1907 and, in 1911, raised him to the highest rank. Real Privy Counsel with the title of Excellency.

Ehrlich married Hedwig Pinkus, then aged 19, in 1883. They had two daughters, Stephanie and Marianne.When the First World War broke out in 1914 it distressed him very much and at Christmas of that year he had a

slight stroke. He recovered quickly, but his health, whieh had never failed him, now began to decline. In 1915 he wentto Bad Homburg for a holiday, and there, on August 20 of that year, he had a second stroke which ended his life.

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Partial Cell Functions