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Further Observations on the Detoxification of Bacterial Toxins*
FURTHER OBSERVATIONS ON THE DETOXIFICATION OF BACTERIAL TOXINS* B y T H O M A S B . HARTZELL, M .D ., D .M .D ., F .A .C .D ., and W . P . L A R SO N , M .D ., M inneapolis, M innesota
E F O R E the etiologic agents o f infection were discovered, practi cally no progress was made in the way o f either the cure or the control o f the infectious diseases. T h e w ork o f Pasteur pointed the way to the solution o f this great problem. Lister was the first to profit by Pasteur’s teaching, in introducing antiseptic surgery, and from that time began the search fo r disinfectants, a search that has con tinued alm ost unabated to the present time. D isinfectants have their place as w ell as their limitations. W e have today numerous agents that quickly destroy bacteria. I t was soon found that most o f the active disinfectants could be used only to a lim ited extent in medical practice, as they were as toxic, in general, to the body as to the micro-organisms. Aside from two or three agents that act specifically, quinin in m alaria, fo r example, we have no drugs which actu ally combat bacterial infection when it exists w ithin the tissue structures. T h e failure adequately to control infections by specific medication led to the search for other means, such as vaccines and specific serums. W e know that it is
B
*Read before the Section on Histology, Physiology, Pathology, Bacteriology and Chemistry (Research) at the Sixty-Seventh Annual Session of the American Dental Asso ciation, Louisville, Ky., Sept. 23, 1925. Jour. A. D. A., August, 1926
now possible to produce an antitoxin against infections where the micro organism w ill secrete its toxin when grow ing in the test tube, as does the diphtheria bacillus. O nly a few bac teria give up their toxin to the lab oratory w orker, and therefore our antitoxins are few in num ber; three, to be exact. In the seventeenth century, a keen physician observed that an attack o f cowpox protected against that terrible scourge smallpox, and he introduced vaccination about as we use it today. Prophylactic vaccination against infec tion in general did not come into use until quite recently. T h is was, of course, because the science o f vaccina tion was not understood even though practiced to some extent. Scientific control o f infections began w ith the work o f Pasteur when he successfully vaccinated aga-inst rabies and anthrax. Pasteur recognized the specificity o f in fections, and the now well understood fact that an attack o f some infections was follow ed by an im m unity to that specific infection, and attem pted to pro duce m ild form s o f infection in order to protect from the more severe forms. As our knowledge o f infections grew, it became recognized that w e had not, as yet, solved the problem o f control, as it was soon observed that many in
1147
1148
The Journal of the American Dental Association
fections could not be controlled by either an attack o f the disease or vacci nation. Pneumococcic and streptococcic infections seem to recur more readily am ong those who have already had one or more attacks. T h e re is, then, one type o f infection that leaves a perm anent im m unity, while, in other types, one attack seems to leave the patient more susceptible than before. I f one examines the list o f diseases that are follow ed by a last ing im m unity, one is struck by the fact that the list is lim ited to those infections caused by the colon-typhoid group. (W e are not including the infections caused by filterable viruses.) Vaccina tion against these diseases has been found to be successful. T h e bacteria o f this group show a peculiar readiness to grow in a medium o f low surface tension. F urtherm ore, these are the bacteria against which antibodies are readily demonstrated. O n the other hand, those bacteria that cause infec tions which are not follow ed by an im m unity are very sensitive to changes in the surface tension o f the medium. Such organisms as the pneumococcus and the streptococcus w ill not grow if the surface tension o f the medium is depressed three or fo u r dynes. In studying this problem w ith this observa tion in m ind, we have found it to be so common as to be w hat is tantam ount to a law. T o state the law concisely: Bacteria that grow w ell in mediums of low surface tension produce infections that are follow ed by a lasting im m u nity, and that may be controlled by artificial vaccination; w hile those bac teria that are sensitive to changes in the surface tension o f the mediums produce infections that are not follow ed by im m unity and that cannot be controlled by artificial im m unization.
T h e most plausible explanation o f this phenomenon is the fact that anti bodies, in order to be effective against their specific bacteria, must be able to attack them. I t has been shown experi m entally by G reenfield and one o f us ( W . P. L .) that bacteria may be so changed in physical composition that they do not readily unite w ith their antibodies. Such bacteria may then be brought back to norm al by cultivation under suitable conditions. Inability o f the bacterium to unite w ith its specific antibody is sometimes spoken o f as the condition o f “ serumfastness.” I t has been observed espe cially w ith the typhoid bacillus that it is o ften serum -fast until a fte r it has been cultivated on artificial mediums for several generations. D uring the course o f the infection, it becomes necessary fo r the m icro-organism to establish itself in such a way as to resist attack by its specific antibodies. T h is factor determines w hether or not an infection is to proceed or to subside. As has been pointed out above, pathogenic bacteria may be divided into two main classes: those that grow w ell in a medium of low surface tension, and those that are unable to develop in a medium the sur face tension o f which has been de pressed two or three dynes below the optimum o f about 60 dynes. T h is view is in harm ony w ith our observation that practically no antibodies may be dem onstrated against the bacteria that are sensitive to surface tension changes, since the antibodies are unable to unite w ith these bacteria. Undoubtedly, be cause o f the physical condition o f the ectoplasm, no antibodies are demon strable; and since the antibodies cannot attack the bacteria, they have no influ ence on their grow th and development. H ere, then, we have an explanation as
H artzell-Larson— Detoxification of Bacterial Toxins to why some infections are follow ed by im m unity, w hile, in other infections, the patient seems to be as sensitive, if not more so, a fte r w ithstanding the in fection. W e have studied the effect o f sur face tension depressants on the patho genicity and toxicity o f some o f the surface tension sensitive organisms, es pecially the streptococcus and pneumo coccus, as w ell as various bacterial toxins. W e have made the interesting observation that the streptococci and pneumococci completely lose their pow ers to in fect a fte r being in contact fo r a few seconds w ith a surface tension depressant such as castor oil soap. W e have found that a 1 per cent solution o f castor oil soap w ill deprive the pneu mococcus and- streptococcus o f their pathogenicity in h a lf a m inute. A ni mals such as w hite mice and rabbits, which are very sensitive to pneum ococcic and streptococcic infections, w ill withstand m any thousand fatal doses a fte r the respective organisms have been treated w ith castor oil soap. T his surface tension depressant not only deprives the bacteria of their in fective properties, but it also neutralizes the toxins, both soluble and endotoxins, .o f the bacteria. D u rin g the past tw o or three years, it has been found that the scarlet fever streptococcus secretes a potent soluble toxin. Some investi gators have found that one one hun dred-thousandth cubic centim eter o f this toxin, when injected into the skin o f an individual, causes an erythema and that one one-thousandth cubic centim eter when injected into an individual, will often cause the typical picture o f scarlet fever. W h en this toxin is mixed with castor oil soap and immediately injected into an individual, we find that one thousand times as much may be injected
1149
w ithout causing any symptoms w h at ever. W e cite this as an example o f the marvelous detoxifying properties o f the castor oil soap. W e have found it practical to detoxify both the bacteria and their toxins by this method fo r pur poses o f im m unization. O ne o f the greatest difficulties in im m unizing animals against the various bacteria and their products, up to the present time, has been the fact that if these antigens were injected in an un modified state, they would cause infec tion and often the death o f the animal. W h en sublethal doses are given fo r purposes o f im m unization, the toxicity o f the antigen alters the power o f the anim al body to react in the way o f anti body form ation, so that im m unization is slow and often impossible. By in jecting the animals with bacterial cul tures that have been detoxified with castor oil soap, we rapidly produce im m unization. W e find it compara tively easy to produce potent serums against both pneumococcic and strep tococcic infections. T h e im m unization is rapid, since tremendous doses may be given w ithout injurious effects; and since no injury follow s the injection, no time is required fo r recuperation, as is the case by the other method. W e find this a practical method o f im m unizing against the various bacterial toxins, such as diphtheritic toxin and scarlet fever toxin. I t has been customary up to the pres ent, in im m unizing against diphtheria, to neutralize the toxin w ith antitoxin; that is, horse serum that contains anti body against the diphtheritic toxin. T h e patients are injected at ten-day in tervals; in all, three or sometimes more injections being given. T h e disadvan tage o f this method lies in the fact that the individual becomes sensitized to
1 ISO
The Journal of the American Dental Association
horse serum, a great liability to a patient in case it should be necessary a t some subsequent date to use antitoxin pre pared from the horse. By detoxifying the diphtheritic toxin w ith castor oil soap, no such sensitization follow s. A fu rth e r advantage is the fact that the im m unization may, as a rule, be accom plished w ith a single injection instead o f three injections, a point o f very great importance in general practice. W h a t has been said o f diphtheritic toxin also applies to the scarlet fever toxin. By detoxifying the toxin with castor oil soap, a thousand times the usual dose may be given and, as a result, im m unization is effected in a few days. W e cite our experience w ith these toxins in order to emphasize the prac ticability o f detoxifying in this way. T h e mechanism o f detoxification is o f great interest. E arly in our work, it seemed that it was closely associated w ith the phenomenon o f surface ten sion, as the m aximum results were ob tained w ithin certain limits w here the surface tension depression was greatest. Extensive investigation on this point has led us to the conclusion that it is an adsorption phenomenon, as the detoxi fication follow s definitely the laws of adsorption. I f a quantity o f charcoal, colloidal soap or starch is added to a m ixture o f soap toxin, we find that the detoxification does not proceed as when these substances have not been added. T h e explanation o f this lies in the fact that the small particles adsorb the soap onto their surfaces, and hence the soap is not free to react w ith the toxin mole cules. T h is and other experiments have led us to the conclusion that the toxin
molecules, which are o f greater dim en sions than the castor oil soap molecules, as evidenced by filtration and dialyzing experiments, adsorb the soap onto their surfaces, and, in that way, become iso lated or imprisoned, and hence are not free to exert their toxic action in the usual way w hen injected into animals. In accordance w ith w ell know physi cal laws, not all o f the toxin would be adsorbed at a given moment, but a small fraction w ill rem ain free, and an equilibrium is thus established between the bound and the free toxin. A s the free toxin is slowly taken up by the body tissues, an equivalent am ount o f the bound toxin is set free in order to m aintain the equilibrium, and, in this way, the toxin is slowly fed to the or ganism at such a rate as not to cause intoxication but rather im m unization. W hen the toxin soap m ixture given has been excessive, it is clear, fro m w hat has just been stated, that there w ill be a larger am ount o f free toxin present and therefore it is possible to intoxicate w ith a so-called neutralized m ixture. T h e principle o f adsorption is one that must be borne in m ind if the castor oil soap is to be used to detoxify bacteria or toxins in the mouth. Because o f its somewhat unpleasant taste, it is not practicable to use a pure solution of castor oil soap. I t is necessary to mix it w ith certain substances that have the ability to cover the taste. T h erefo re, in making such a preparation, one must bear in mind the necessity o f having the castor oil soap in such proportions that it w ill not all be adsorbed onto the ingredients with which it may be mixed. T h e re must always be some soap in excess o f w hat would be adsorbed onto the surfaces o f such substances.
E F O R E the etiologic agents o f infection were discovered, practi cally no progress was made in the way o f either the cure or the control o f the infectious diseases. T h e w ork o f Pasteur pointed the way to the solution o f this great problem. Lister was the first to profit by Pasteur’s teaching, in introducing antiseptic surgery, and from that time began the search fo r disinfectants, a search that has con tinued alm ost unabated to the present time. D isinfectants have their place as w ell as their limitations. W e have today numerous agents that quickly destroy bacteria. I t was soon found that most o f the active disinfectants could be used only to a lim ited extent in medical practice, as they were as toxic, in general, to the body as to the micro-organisms. Aside from two or three agents that act specifically, quinin in m alaria, fo r example, we have no drugs which actu ally combat bacterial infection when it exists w ithin the tissue structures. T h e failure adequately to control infections by specific medication led to the search for other means, such as vaccines and specific serums. W e know that it is
B
*Read before the Section on Histology, Physiology, Pathology, Bacteriology and Chemistry (Research) at the Sixty-Seventh Annual Session of the American Dental Asso ciation, Louisville, Ky., Sept. 23, 1925. Jour. A. D. A., August, 1926
now possible to produce an antitoxin against infections where the micro organism w ill secrete its toxin when grow ing in the test tube, as does the diphtheria bacillus. O nly a few bac teria give up their toxin to the lab oratory w orker, and therefore our antitoxins are few in num ber; three, to be exact. In the seventeenth century, a keen physician observed that an attack o f cowpox protected against that terrible scourge smallpox, and he introduced vaccination about as we use it today. Prophylactic vaccination against infec tion in general did not come into use until quite recently. T h is was, of course, because the science o f vaccina tion was not understood even though practiced to some extent. Scientific control o f infections began w ith the work o f Pasteur when he successfully vaccinated aga-inst rabies and anthrax. Pasteur recognized the specificity o f in fections, and the now well understood fact that an attack o f some infections was follow ed by an im m unity to that specific infection, and attem pted to pro duce m ild form s o f infection in order to protect from the more severe forms. As our knowledge o f infections grew, it became recognized that w e had not, as yet, solved the problem o f control, as it was soon observed that many in
1147
1148
The Journal of the American Dental Association
fections could not be controlled by either an attack o f the disease or vacci nation. Pneumococcic and streptococcic infections seem to recur more readily am ong those who have already had one or more attacks. T h e re is, then, one type o f infection that leaves a perm anent im m unity, while, in other types, one attack seems to leave the patient more susceptible than before. I f one examines the list o f diseases that are follow ed by a last ing im m unity, one is struck by the fact that the list is lim ited to those infections caused by the colon-typhoid group. (W e are not including the infections caused by filterable viruses.) Vaccina tion against these diseases has been found to be successful. T h e bacteria o f this group show a peculiar readiness to grow in a medium o f low surface tension. F urtherm ore, these are the bacteria against which antibodies are readily demonstrated. O n the other hand, those bacteria that cause infec tions which are not follow ed by an im m unity are very sensitive to changes in the surface tension o f the medium. Such organisms as the pneumococcus and the streptococcus w ill not grow if the surface tension o f the medium is depressed three or fo u r dynes. In studying this problem w ith this observa tion in m ind, we have found it to be so common as to be w hat is tantam ount to a law. T o state the law concisely: Bacteria that grow w ell in mediums of low surface tension produce infections that are follow ed by a lasting im m u nity, and that may be controlled by artificial vaccination; w hile those bac teria that are sensitive to changes in the surface tension o f the mediums produce infections that are not follow ed by im m unity and that cannot be controlled by artificial im m unization.
T h e most plausible explanation o f this phenomenon is the fact that anti bodies, in order to be effective against their specific bacteria, must be able to attack them. I t has been shown experi m entally by G reenfield and one o f us ( W . P. L .) that bacteria may be so changed in physical composition that they do not readily unite w ith their antibodies. Such bacteria may then be brought back to norm al by cultivation under suitable conditions. Inability o f the bacterium to unite w ith its specific antibody is sometimes spoken o f as the condition o f “ serumfastness.” I t has been observed espe cially w ith the typhoid bacillus that it is o ften serum -fast until a fte r it has been cultivated on artificial mediums for several generations. D uring the course o f the infection, it becomes necessary fo r the m icro-organism to establish itself in such a way as to resist attack by its specific antibodies. T h is factor determines w hether or not an infection is to proceed or to subside. As has been pointed out above, pathogenic bacteria may be divided into two main classes: those that grow w ell in a medium of low surface tension, and those that are unable to develop in a medium the sur face tension o f which has been de pressed two or three dynes below the optimum o f about 60 dynes. T h is view is in harm ony w ith our observation that practically no antibodies may be dem onstrated against the bacteria that are sensitive to surface tension changes, since the antibodies are unable to unite w ith these bacteria. Undoubtedly, be cause o f the physical condition o f the ectoplasm, no antibodies are demon strable; and since the antibodies cannot attack the bacteria, they have no influ ence on their grow th and development. H ere, then, we have an explanation as
H artzell-Larson— Detoxification of Bacterial Toxins to why some infections are follow ed by im m unity, w hile, in other infections, the patient seems to be as sensitive, if not more so, a fte r w ithstanding the in fection. W e have studied the effect o f sur face tension depressants on the patho genicity and toxicity o f some o f the surface tension sensitive organisms, es pecially the streptococcus and pneumo coccus, as w ell as various bacterial toxins. W e have made the interesting observation that the streptococci and pneumococci completely lose their pow ers to in fect a fte r being in contact fo r a few seconds w ith a surface tension depressant such as castor oil soap. W e have found that a 1 per cent solution o f castor oil soap w ill deprive the pneu mococcus and- streptococcus o f their pathogenicity in h a lf a m inute. A ni mals such as w hite mice and rabbits, which are very sensitive to pneum ococcic and streptococcic infections, w ill withstand m any thousand fatal doses a fte r the respective organisms have been treated w ith castor oil soap. T his surface tension depressant not only deprives the bacteria of their in fective properties, but it also neutralizes the toxins, both soluble and endotoxins, .o f the bacteria. D u rin g the past tw o or three years, it has been found that the scarlet fever streptococcus secretes a potent soluble toxin. Some investi gators have found that one one hun dred-thousandth cubic centim eter o f this toxin, when injected into the skin o f an individual, causes an erythema and that one one-thousandth cubic centim eter when injected into an individual, will often cause the typical picture o f scarlet fever. W h en this toxin is mixed with castor oil soap and immediately injected into an individual, we find that one thousand times as much may be injected
1149
w ithout causing any symptoms w h at ever. W e cite this as an example o f the marvelous detoxifying properties o f the castor oil soap. W e have found it practical to detoxify both the bacteria and their toxins by this method fo r pur poses o f im m unization. O ne o f the greatest difficulties in im m unizing animals against the various bacteria and their products, up to the present time, has been the fact that if these antigens were injected in an un modified state, they would cause infec tion and often the death o f the animal. W h en sublethal doses are given fo r purposes o f im m unization, the toxicity o f the antigen alters the power o f the anim al body to react in the way o f anti body form ation, so that im m unization is slow and often impossible. By in jecting the animals with bacterial cul tures that have been detoxified with castor oil soap, we rapidly produce im m unization. W e find it compara tively easy to produce potent serums against both pneumococcic and strep tococcic infections. T h e im m unization is rapid, since tremendous doses may be given w ithout injurious effects; and since no injury follow s the injection, no time is required fo r recuperation, as is the case by the other method. W e find this a practical method o f im m unizing against the various bacterial toxins, such as diphtheritic toxin and scarlet fever toxin. I t has been customary up to the pres ent, in im m unizing against diphtheria, to neutralize the toxin w ith antitoxin; that is, horse serum that contains anti body against the diphtheritic toxin. T h e patients are injected at ten-day in tervals; in all, three or sometimes more injections being given. T h e disadvan tage o f this method lies in the fact that the individual becomes sensitized to
1 ISO
The Journal of the American Dental Association
horse serum, a great liability to a patient in case it should be necessary a t some subsequent date to use antitoxin pre pared from the horse. By detoxifying the diphtheritic toxin w ith castor oil soap, no such sensitization follow s. A fu rth e r advantage is the fact that the im m unization may, as a rule, be accom plished w ith a single injection instead o f three injections, a point o f very great importance in general practice. W h a t has been said o f diphtheritic toxin also applies to the scarlet fever toxin. By detoxifying the toxin with castor oil soap, a thousand times the usual dose may be given and, as a result, im m unization is effected in a few days. W e cite our experience w ith these toxins in order to emphasize the prac ticability o f detoxifying in this way. T h e mechanism o f detoxification is o f great interest. E arly in our work, it seemed that it was closely associated w ith the phenomenon o f surface ten sion, as the m aximum results were ob tained w ithin certain limits w here the surface tension depression was greatest. Extensive investigation on this point has led us to the conclusion that it is an adsorption phenomenon, as the detoxi fication follow s definitely the laws of adsorption. I f a quantity o f charcoal, colloidal soap or starch is added to a m ixture o f soap toxin, we find that the detoxification does not proceed as when these substances have not been added. T h e explanation o f this lies in the fact that the small particles adsorb the soap onto their surfaces, and hence the soap is not free to react w ith the toxin mole cules. T h is and other experiments have led us to the conclusion that the toxin
molecules, which are o f greater dim en sions than the castor oil soap molecules, as evidenced by filtration and dialyzing experiments, adsorb the soap onto their surfaces, and, in that way, become iso lated or imprisoned, and hence are not free to exert their toxic action in the usual way w hen injected into animals. In accordance w ith w ell know physi cal laws, not all o f the toxin would be adsorbed at a given moment, but a small fraction w ill rem ain free, and an equilibrium is thus established between the bound and the free toxin. A s the free toxin is slowly taken up by the body tissues, an equivalent am ount o f the bound toxin is set free in order to m aintain the equilibrium, and, in this way, the toxin is slowly fed to the or ganism at such a rate as not to cause intoxication but rather im m unization. W hen the toxin soap m ixture given has been excessive, it is clear, fro m w hat has just been stated, that there w ill be a larger am ount o f free toxin present and therefore it is possible to intoxicate w ith a so-called neutralized m ixture. T h e principle o f adsorption is one that must be borne in m ind if the castor oil soap is to be used to detoxify bacteria or toxins in the mouth. Because o f its somewhat unpleasant taste, it is not practicable to use a pure solution of castor oil soap. I t is necessary to mix it w ith certain substances that have the ability to cover the taste. T h erefo re, in making such a preparation, one must bear in mind the necessity o f having the castor oil soap in such proportions that it w ill not all be adsorbed onto the ingredients with which it may be mixed. T h e re must always be some soap in excess o f w hat would be adsorbed onto the surfaces o f such substances.