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Bacteriological Studies of Multiple Sclerosis
Published in Annals of Allergy, Vol. 6, pp. 271-292. May-June, 1948.
* * *
The clinical diagnosis of the patients in this study was made by attending physicians and by consulting neurologists. The patients studied represented samplings of the disease from widely separated regions and widely different climates. The clinical history and physical examination gave no clue as to the probable cause of the disease, and its progressive course was similar regardless of place of residence, climate, occupation or nationality. The isolation, from infection atria in persons suffering from multiple sclerosis, of streptococci having elective affinity for the nervous system of animals and neurotropic cataphoretic velocity has been reported.9,10 Evidence for the possible etiologic relationship of alpha streptococci isolated chiefly from nasopharynx, tonsils and teeth was sought by appropriate inoculation of animals, by agglutination and precipitation tests with the serums from persons afflicted, with anti-streptococcic serum prepared in horses,11 and with thermal antibody prepared in vitro from the streptococcus in NaCl solution suspensions by prolonged heating in the autoclave,12 and with much less heat plus hydrogen peroxide.14 In addition, presumptive tests were made for specific streptococcal antigen and antibody in skin or blood by intracutaneous injection of natural and thermal antibody and of streptococcal antigen, respectively.15
Methods of Study
Pus or other exudate was expressed and scooped from infected tonsils with a small-sized laryngeal mirror bent to an angle of 35°. Pus was aspirated from the depths of pyorrhea pockets with a capillary pipette. Pulpless teeth were drawn in a sterile manner and the apical end was immediately severed with a bone-cutting forceps. The nasopharynx was swabbed without touching the tongue, with aluminum wire cotton-wrapped swabs bent to a suitable angle to obtain material from up and behind the soft palate. The exudates from sinuses in persons having sinusitis, from cervix uteri in women suffering from endocervicitis, from the prostate in men having prostatitis and from the stool in special instances were obtained by suitable means.
The several materials thus obtained were suspended routinely in 2 ml. of 0.2 per cent gelatine in Locke’s solution, for microscopic examination of Gram-safranin stained films, for inoculation of animals and for cultures. The type and dosage for direct inoculation of animals and for cultures was modified as indicated by the kind and number of bacteria found in stained films. As a rule, two rabbits were inoculated intracerebrally far forward in the right frontal lobe, one with 0.1 ml, the other with 0.2 ml. of the suspension in 2 ml. of gelatine-Locke’s solution of the washings of nasopharyngeal swabbing, of washing of the apices of pulpless teeth, and of pus from pyorrhea pockets and tonsils. Cultures of these were made on blood agar and in dextrose brain broth. The latter afforded a gradient of oxygen tension and other conditions which were found essential for the growth and isolation of specific types of alpha streptococci.
The dextrose brain broth was either freshly prepared or was boiled to drive off oxygen and cooled immediately before being inoculated. Seven ml. of blood were drawn into vacuum tubes, allowed to clot, centrifuged, the serum poured off, the clot partially macerated and planted into dextrose brain broth. Cultures in this medium were also made of the freshly drawn spinal fluid. The dextrose brain broth was prepared by adding pieces of fresh or frozen calf or young beef brain to tall columns, 9 to 10 cm. in test tubes of 0.2 per cent dextrose broth adjusted to pH 7.2, in a proportion of approximately one part by volume of brain substance to seven parts of the dextrose broth, before autoclaving at 17 pounds pressure for thirty minutes. All cultures were incubated at 33° to 35° C.
Early in these studies, animals were inoculated with the primary cultures from the one tube of dextrose brain broth which had been inoculated, provided stained films revealed a pure culture of the streptococcus. Blood agar plates were made at the same time to determine the purity and type of streptococcus. If other organisms had also grown in the dextrose brain broth, blood agar plates were made, and the subculture in dextrose brain broth from one or more colonies of the streptococcus which grew on the blood agar plate was injected into animals. More recently it was found that specifically virulent streptococci grew in far higher serial dilutions in dextrose brain broth than saprophytic variants and other bacteria often also present in the material studied. Serial dilutions were made in dextrose brain broth, at steps of 1-100 or 1-10,000 in four to six or more tubes, each containing 15 ml. of this medium. Serial dilutions at 1-100 were made by transferring and thoroughly mixing 0.15 ml. with the same 1 ml. pipette from tube to tube, and dilutions at 1-10,000 were made with a nichrome wire which was not sterilized after the first transfer and to which there adhered approximately 1.5 cubic millimeters of the culture. Pure cultures of the streptococcus for inoculation of animals and other studies were obtained from the end point of growth. Rabbits and guinea pigs were routinely inoculated intracerebrally with 0.1 or 0.2 ml. of a 1-200 or 1-10,000 dilution in NaCl solution, and mice with 0.03 ml. of the dextrose brain broth culture, and with these amounts, respectively, of 1-10,000 dilution of pure cultures in the autoclaved chick embryo medium.13 Pure cultures of the streptococcus thus isolated in dextrose brain broth from the several materials from persons and from the brain or blood of inoculated animals were grown for one culture generation in varying amounts of 0.2 per cent dextrose broth. These cultures were centrifuged, the supernatant liquid was discarded, and the streptococci were placed in dense suspensions of glycerol, two parts, and saturated NaCl solution, one part. These dense suspensions were made to contain the growth of from 150 to 500 ml. of the culture or approximately 300,000,000,000 to 1,000,000,000,000 organisms per ml. The suspensions containing the freshly isolated streptococci were kept in the refrigerator, and appropriate dilutions were made for precipitation and agglutination tests, for immunization of horses and for the preparation of vaccines and thermal antibodies. Agglutination tests were made at twofold dilutions of 1-20 to 1-160 or fivefold dilution of 1-10 to 1-1,250 of the serum from patients, and at fivefold dilutions of 1-20 to 1-2,500 of anti-streptoccic serums and thermal antibody, in saline containing 0.2 per cent phenol, against suspensions in saline containing 0.2 per cent phenol and approximately 3,000,000,000 streptococci per ml. in the final dilution. The mixtures were incubated at from 48° to 50°C. for eighteen to twenty-four hours when readings were made. The degree of agglutination in each of the four dilutions was recorded according to the scale of 0 to 4 plus. A 4-plus agglutination in each dilution, or 16, would represent 100 per cent; a total of 6-plus, or 6/16 equalled 37.5 per cent of the total possible agglutination. The brain of animals and pieces of the medulla and spinal cord were fixed in 10 per cent formalin. Sections were stained with hematoxylin and eosin, by the Weigert method for myelin, and by a modified Gram-Weigert stain for bacteria, in which decolorization was carried to a fair blue instead of to the end point, and no counter stain was used.
Results
Cultures from the blood and spinal fluid in dextrose brain broth proved negative except in a few instances in which alpha type of streptococci were isolated. Intracerebral injections of the spinal fluid in animals were without effect. Cultures from material obtained from nasopharynx, tonsils and infected teeth uniformly yielded streptococci of the viridans or alpha type, in great preponderance or in pure culture, and only occasionally small numbers of beta hemolytic streptococci. Staphylococci and micrococcus catarrhalis often grew in varying numbers on the primary blood agar plate, and H. influenzae almost never.
A careful search for associated infections was made in thirty-two cases. A history of repeated attacks of tonsillitis was obtained in seventeen, of an antecedent attack of influenza in thirteen. Clearly infected tonsils or tonsilar tags were found in nineteen, pyorrhea in twenty-four, and one or more pulpless teeth in twenty-eight.17 In no instance did the disease begin following removal of foci of infection nor could relapses be traced to the removal. Since the methods and period of time covered in studies of multiple sclerosis were similar to those made of other diseases of the nervous system, and to afford a ready means of comparing the results obtained, a summary of the results in these and in normal controls is included.
The mortality, incidence of symptoms, and isolations of the streptococcus from the brain of rabbits that were inoculated directly with saline suspensions or with cultures of the streptococcus on isolation and after animal-passage, from altogether sixty-one persons suffering from multiple sclerosis, and in contrast the result in rabbits similarly inoculated with streptococci from spasmodic torticolis, persistent epidemic hiccup, encephalitis, poliomyelitis, epilepsy and schizophrenia and normal controls, are summarized in Table I. The mortality and isolations of streptococci from brain of inoculated rabbits and the incidence of symptoms referable to the nervous system were consistently much higher following inoculations of streptococci from persons suffering from multiple sclerosis and the other diseases of the nervous system than in those receiving the streptococcus from normal controls. The incidence and type of symptoms referable to the nervous system in the three groups of rabbits receiving streptococci isolated in studies of multiple sclerosis were strikingly similar to those at hand in persons suffering from this disease. The mortality and incidence of isolations of the streptococcus from brain of mice and guinea pigs receiving the streptococcus from multiple sclerosis were similar to those obtained in rabbits. Lesions of lungs in mice inoculated intraperitoneally were abnormally high, occurring in ten of eighteen mice so inoculated. Of 117 mice inoculated intracerebrally with forty strains of the streptococcus, seventy-three (62 per cent) died. Severe tremors were observed in forty-five, spasms in twenty-eight, ataxia in ten, paralysis in twenty-three and incontinence of urine in six. The streptococcus was isolated from the brain in thirty-four of the thirty-eight cultured. Of sixty-eight guinea pigs inoculated intracerebrally with twenty-six strains, thirty-six (53 per cent) died. Severe tremors developed in thirty-one, spasms in eighteen, ataxia in ten, paralysis in twenty-five, incontinence of urine in four and salivation with wet fur under chin in five. The streptococcus was isolated from the brain in twenty-seven of the twenty-nine cultured.
Table I. Mortality and Symptoms in Rabbits Following Intracerebral Inoculation of Streptococci Isolated in Studies of Multiple Sclerosis and Other Diseases of the Nervous System and Isolation of Streptococci from the Brain of Inoculated Animals
*Usually of severe “intension” type.
**Accompanied by tic-like and spasmodic movements of the head.
***Especially of the diaphragm.
The incidence and type of symptoms in animals receiving the streptococcus isolated in studies of the other diseases of the nervous system were likewise similar in important respects to those more or less characteristic of the diseases in question. The statistical evidence of specificity, though often striking, does not adequately represent the results. Blurred vision or blindness, hyperactive reflexes and incontinence of urine, which developed not uncommonly in animals following inoculation of streptococci isolated in studies of multiple sclerosis, were not obtained or were obtained less often following inoculation of streptococci isolated in studies of the other diseases, and never following inoculation of the streptococcus from normal controls. Paralysis in the multiple sclerosis group of animals was relatively mild, usually spastic in type and was often associated with localized spasms. Ataxia, accompanied by tic-like and spasmodic movements of the head, was the characteristic picture in animals following inoculation of the streptococcus isolated in studies of spasmodic torticolis. Ataxia spasms of the diaphragm and abdominal muscles, sometimes associated with audible hiccup and hemorrhages in the diaphragm, characterized the findings in animals inoculated with the streptococcus isolated in studies of epidemic and postoperative hiccup. A wide range of symptoms developed in rabbits receiving the streptococcus isolated in studies of encephalitis, which corresponded to the wide range of symptoms at hand in the patients from whom the streptococcus inoculated was obtained. Muscular spasms and lethargy occurred in high incidence in animals receiving the streptococcus isolated, respectively, from persons having myoclonic and lethargic types of encephalitis. Flaccid paralysis with diminution or loss of knee jerks occurred in highest incidence in the poliomyelitis group of animals, and tremors, spasms, and ataxia were slight or absent. Moreover, nystagmus and incontinence of urine almost never occurred. The presence of specific types of streptococci in epidemic encephalitis and poliomyelitis is obviously not to be considered as the sole cause of these diseases independently of the viruses. Severe tremors and spasms, often associated with generalized convulsions resembling grand mal, occurred in highest incidence in the group of animals receiving the streptococcus from persons suffering from idiopathic epilepsy.16 Extreme hyperirritability and tremors occurred in highest incidence in the group of animals receiving the streptococcus isolated in studies of schizophrenia. Catatonic states and strange changes in behavior often also developed in these, and virtually never occurred in the other groups of animals.16
Sterile filtrates of NaCl solution suspensions of material obtained directly from nasopharynx, tonsils and teeth, and of the dextrose brain broth cultures from persons having multiple sclerosis, when injected intracerebrally, caused transient tremors, in-coordination, congestion of eyes, and other mild symptoms soon after injection, followed by recovery. Late symptoms or deaths, indicating the possible presence of a virus, did not occur.
Streptococci isolated from the cervix uteri, from prostate and from the stool were without neurotropic virulence. The invasiveness or general virulence of the streptococcus isolated in studies of multiple sclerosis, in accord with the nature of the disease, was found to be of a low order. Inoculated animals did not die of a streptococcemia. Cultures were made of the blood of ninety-two rabbits that died after intracerebral inoculation of the streptococcus. The cultures remained sterile in seventy-three, and in only nineteen was the streptococcus obtained. Of the nineteen rabbits, the streptococcus was isolated in nine of twelve that died in twenty-four hours after inoculation; in six of nineteen that died on the second day; in three of thirteen that died on the third day; in one of eight that died on the fourth day, and in none of forty that died on the fifth to the sixtieth day.
As shown in Table I, cultures were made from the brain in 104 of the 122 rabbits that had been inoculated with material containing the streptococcus. Sixty-five (63 per cent) yielded the streptococcus. While the cultures in thirty-nine proved sterile, most of the thirty-nine died late from the effects of the inoculation, and a few were anesthetized while having active symptoms. Cultures from the blood of all of these remained sterile, and the streptococcus was not demonstrable in the lesions. The late symptoms and deaths were clearly due to causes other than an overwhelming infection. The probable presence of a streptococcal neurotoxin having predilection for vital nerve centers, or to which the vital centers had become allergic, was considered. Accordingly, emulsions of the brain and filtrates of emulsions were made from rabbits that died late, and whose blood and brain proved sterile, and from normal rabbits. These were inoculated intracerebrally into normal rabbits. All of six rabbits receiving emulsions and four receiving filtrates died —three on the day after inoculation, two in three days, two in four days, one in seven, and two in fourteen days. The material inoculated and cultures from the brain of all that died proved sterile. None of these rabbits developed symptoms suggestive of multiple sclerosis. The emulsions of the brain and filtrates of emulsions from normal rabbits similarly inoculated proved innocuous.
Illustrative Experiments in Animals and Protocols
A mixture of equal parts of the cultures of the streptococcus isolated from the nasopharynx of sixteen persons suffering from multiple sclerosis, and grown separately in chick embryo medium for from seven to forty-two days, was made, and a 1-10,000 dilution was inoculated intracerebrally in eight rabbits, twelve guinea pigs and one monkey. Intension tremors developed in seven of the eight rabbits and in eight of the twelve guinea pigs, ataxia in six and five, respectively, exaggerated knee jerks in eight and seven, nystagmus in six and two, ataxia in six and two, localized paralysis in seven and eight, spasms of muscles in five and six, incontinence of urine in four and three, and drooling of saliva in two and four. Blurred vision developed in three, and blindness in two, of the eight rabbits. Seven of the rabbits and seven of the guinea pigs died in from two to twelve days following inoculation. The rest were anesthetized. The streptococcus was isolated from the brain in, dextrose brain broth in all of the twelve guinea pigs and in the rabbits that died within six days, and in only one of the rest. The details of experiments and results obtained in this series of rabbits, guinea pigs and the monkey, and in other rabbits, are depicted in Protocols 1, 2, 3, and 4.
Protocol 1.—A white rabbit weighing 1,800 grams was injected intracerebrally on June 11, 1943, with 0.1 ml. of sterile chick embryo medium to lower the inherently high resistance of the brain to infection, and 2 ml. of the undiluted culture from nasopharynx was injected into the tongue. The animal was well June 12. On June 13, moderate tremors of masseters and muscles of the neck and tremors of extremities on exertion were noted. On June 14, weakness of the left fore extremity, severe tremors and twitchings of the muscles of the neck, exaggerated reflexes and ataxia had developed. The paralysis and tremors were worse on June 15, and the animal was found dead on June 16. Moderate hemorrhagic edema of lungs and congestion of the brain were found. Cultures in dextrose brain broth from the brain and blood yielded a pure culture of the streptococcus.
Protocol 2.—A medium-sized white rabbit was inoculated intracerebrally on June 1, 1943, with 0.1 ml. of a 1-10,000 dilution in 0.2 per cent dextrose brain agar of a serial dilution culture of the streptococcus which grew in dextrose brain broth at 10-8 dilution of the NaCl solution washings of the nasopharyngeal, swabbing. On June 2 the animal seemed well when quiet in its cage, but on exertion, tremors of extremities developed. On June 3, extreme intension tremors, ataxia, exaggerated knee reflexes and weakness of adductors of fore extremities were noted. From June 3 to 8, the animal remained about the same. On June 9 it seemed well when quiet in cage, but on exertion, severe tremors, ataxia, blurred vision, horizontal nystagmus and tilting of head to the right were noted. On June 10 it was about the same. On June 11 and 12, the symptoms were worse, and the animal was found dead on June 13. There was no mark at the point of intracerebral injection. The meninges were normal. The brain was moderately congested. The optic nerves were congested and edematous. There were no lesions of the viscera. Dextros brain broth cultures from the brain yielded a pure culture of the streptococcus.
Protocol 3.—A guinea pig weighing 350 grams was inoculated intracerebrally on June 11, 1943, with 0.01 ml. of a 1-10,000 dilution of the chick embryo cultures of the streptococcus. On June 12 and 13, there were no apparent symptoms. On June 14 severe tremors on exertion and moderate spastic paralysis of hind extremities were noted. On June, 16 spastic gait, ataxia, severe intension tremors, nystagmus and wetting of the fur under the chin were present. On June 17 the symptoms were about the same. On June 19 intension tremors were extreme, often bordering on generalized spasms, associated with severe ataxia and spastic weakness of hind extremities. It was anesthetized to death. Fur under the chin was wet due to drooling of saliva. Aside from moderate congestion of the brain, no lesions were found. Dextrose brain broth cultures from the brain yielded a pure culture of the streptococcus.
Protocol 4.—A medium-sized rhesus monkey was inoculated intracerebrally on June 1, 1943, with 2 ml. of a 1-10,000 dilution in 2 per cent dextrose brain agar of the primary dextrose brain broth culture of the streptococcus from the nasopharyngeal swabbing of a person in the active stage of multiple sclerosis. The monkey was apparently well on June 2. On June 3 it seemed well when undisturbed in its cage, but when it jumped from its cage, undoubted weakness of hind extremities was noted. On June 4, severe tremors on exertion, horizontal nystagmus and incontinence of urine had developed. The temperature was normal. These symptoms disappeared, and on June 11 it was inoculated intracerebrally with 1 ml. of a 1-10,000 dilution of the mixtures of the chick embryo cultures of the streptococci from the sixteen persons having active symptoms of multiple sclerosis, and intralingually with 2 ml. of the undiluted mixture of cultures. On June 12 at 9:00 a.m., the animal refused to leave its cage, and when made to do so, severe tremors and undoubted weakness of hind extremities and incontinence of urine became manifest, and exaggerated knee jerks were elicited. On June 13 it sat quietly in its cage, apparently blind. On exertion, severe tremors, bordering on mild generalized spasms, ataxia, and spastic weakness of extremities were noted. It bumped into the walls of its cage as it moved aimlessly about. The pupils were widely dilated and did not respond to light. Severe and continuous horizontal nystagmus had developed. On June 14 it was found dead. Necropsy revealed congestion of the brain, infiltration and edema surrounding the optic nerve and chiasm, and also of spinal nerves and the anterior aspect of the medulla. A small cyst was found at the point of injection in the right frontal lobe. The viscera were normal. Dextrose brain broth cultures of pipettings of cerebrospinal fluid admixed with brain substance revealed a pure culture of the streptococcus.
Results of Agglutination and Precipitation Tests
The results of agglutination tests made with the serums and streptococci from persons having multiple sclerosis, in contrast to those obtained in control studies, are summarized in Table II. It will be seen that the streptococci isolated in studies of multiple sclerosis, migraine, schizophrenia and arthritis were agglutinated in highest titer by the respective homologous serums. Moreover, the agglutinative titer of the serums and the antibody titer in skin or blood, as determined by the cutaneous tests, ran closely parallel. This was especially true in multiple sclerosis. The streptococci isolated during the quiescent stage of multiple sclerosis were not agglutinated by any of the serums.
Table II. Agglutinative Titer of the Serum of Persons Suffering from Multiple Sclerosis, Migraine, Schizophrenia and Arthritis for Streptococci Isolated in Studies of the Respective Diseases
The agglutinative titers of serums from persons having active multiple sclerosis, and, in contrast, of serums from persons convalescing from respiratory infections for streptococci isolated in studies of persons having these and other diseases, are summarized in Table III. A high degree of respective specificity is shown.
Table III. Agglutination of Streptococci Isolated in Studies of Multiple Sclerosis and Other Diseases by the Serums of Persons Having Multiple Sclerosis and the Serums from Persons Convalescing from Respiratory Infections
The results of a long series of agglutinative tests with antiserums prepared in horses with streptococci isolated in studies of different diseases of the nervous system, of influenza and of arthritis, and the homologous, closely and distantly related streptococci, are summarized in Table IV. Agglutinative titers for homologous and closely related strains were consistently much higher than for more distantly related strains. Evidence indicating antigenic and other differences in alpha streptococci isolated in studies of multiple sclerosis, and of those isolated in studies of other diseases, is strikingly shown in Table V. Comparable suspensions in NaCl solution of each of the different groups of strains, when autoclaved with hydrogen peroxide, yielded agglutinins in highest titer for the respective homologous strains.14
Table IV. Agglutinative Titer of Anti Streptococcic Serums for Closely Related, Homologous and Distantly Related Streptococci
Table V. Agglutination of Streptococci by Thermal Antibody Prepared from Streptococci Isolated in Studies of Multiple Sclerosis and Other Diseases
Results of precipitation reactions at the interface between the serums of horses that had been immunized with streptococci from encephalitis, poliomyelitis and respiratory infections closely related to streptococci from multiple sclerosis, and the serums and NaCI solution extracts of nasopharyngeal swabbings from persons having multiple sclerosis, are summarized in Table VI. A much higher incidence of precipitation occurred with the antiserums from encephalitis, poliomyelitis and respiratory infections than with antiserums prepared with the streptococci from arthritis, and than with anti-pneumococcic and normal horse serum.
Table VI. Precipitation at the Interface Between the Serums and Washings of Nasopharyngeal Swabbings of Persons Having Multiple Sclerosis and Antiserums Prepared with Streptococci Isolated in Studies of Encephalitis, Poliomyelitis and Respiratory Infections
The effects of intravenous injections of histamine on the specific streptococcal antigen content in skin or blood, determined by the intradermal injection of “natural” and artificial or thermal streptococcal antibody12 in persons having multiple sclerosis, are summarized in Table VII. A significant diminution in antigen occurred, as measured by the intradermal injection of natural antibody prepared in horses with streptococci isolated in studies of encephalitis and poliomyelitis, and a striking specific drop occurred as measured with thermal antibody prepared in vitro from streptococci isolated in studies of multiple sclerosis.
Table VII. Erythematous Reactions Following Intradermal Injection of “Natural” and Thermal Antibody in Persons Having Multiple Sclerosis in Relation to Histamine Injections
The effects of intravenous injections of histamine on the cutaneous reactions to the specific thermal antibody and antigen in thirteen persons having multiple sclerosis are summarized in Table VIII. The reactions to antibody, indicating antigen, were uniformly far greater, and to antigen, indicating antibody in skin or blood, were uniformly far less, in the two persons before treatment with histamine and the seven other persons not receiving histamine (not included in the table) than the reactions indicating antigen and antibody, respectively, one-half to two hours after daily intravenous histamine injections. Moreover, reactions indicating antigen were significantly less in twelve of the thirteen persons receiving histamine, and reactions indicating antibody greater in eight, one-half to two hours after histamine, than the reactions twenty-four hours after histamine injections. The diminution in antigen and increase in antibody was by far the greatest after the first histamine treatment.
Table VIII. The Effect of Histamine Injections in Persons Having Multiple Sclerosis on the Specific Streptococcal Antigen and Antibody Titer in Skin or Blood as Determined by the Reaction Following Intracutaneous Injection Respectively of Thermal Antibody and of Antigen
The effects on the cutaneous reactions to natural antibody, prepared with closely related streptococci from encephalitis and poliomyelitis, of treatment of persons having multiple sclerosis with histamine alone, and of treatment with histamine and specific thermal antibody, are summarized in Table IX. It will be seen that there was a far greater reduction in reactions indicating antigen following the combined treatment, than with histamine alone. This is in accord with the results obtained in a number of persons having multiple sclerosis treated with vaccine and thermal antibody prepared from streptococci isolated in studies of multiple sclerosis. These results were similar to those obtained in a man of middle age having advanced multiple sclerosis. The cutaneous reaction to antibody on January 3, 1947, before treatment, was 19.64 sq. cm., and to antigen, 0. On February 24 these reactions were 15.90 and 12.57, respectively; on May 21, 7.07 and 4.91, and on Nov. 11, 3.14 and 4.91. Coincident with the striking reduction of antigen and increase of antibody, the symptoms indicating activity disappeared.
Table IX. The Effect on Cutaneous Reactions in Persons Having Multiple Sclerosis of Treatment with Histamine Alone and Treatment with Histamine and Specific Thermal Antibody
*Thermal antibody was injected subcutaneously every other or every third day. Each injection consisted of 1 ml. of a 1-10 dilution of the supernatant of NaCl solution suspension of 20,000,000,000 streptococci per ml. isolated in studies of multiple sclerosis after autoclaving for 96 hours.
The Gross and Microscopic Lesions
Abscess formation in the brain at the site of injection of material containing the streptococcus and diffuse suppurative meningitis almost never occurred. Diffuse congestion of the brain, edema, and leukocytic infiltration over the anterior surface of the pons and medulla were common, especially in rabbits that died in from two to four days following inoculation of washings of nasopharyngeal swabbings and suspensions of pus from tonsils and pyorrhea pockets. This was less common following inoculation of highly dilute pure cultures of the streptococci. Severe congestion of the mucous membrane of the trachea, with or without hemorrhagic edema of the lungs, was found commonly in rabbits that succumbed soon after inoculation. The bladder was often greatly distended with urine in animals in which severe paralysis had developed.
The microscopic lesions were most numerous in the white matter of the cerebrum, pons, peduncles of cerebellum and the posterior and lateral columns of the spinal cord, Hemorrhages, edema, polymorphonuclear leukocytic infiltration, and degeneration, especially surrounding blood vessels, characterized the microscopic picture in animals that succumbed soon after inoculation. Degeneration and infiltration by lymphocytes and plasma cells predominated in the lesions of animals that died or were anesthetized long after inoculation. The lesions were often related to blood vessels which were partially or completely occluded by thrombi, or more often, by endovascular and perivascular proliferation of cells resembling lymphocytes, endothelial, and plasma cells (Figs. 1, 2 and 3). Occlusion of blood vessels by fibrinous clots of fibrin were not found.6 Hemorrhage and edema were found in the wall and immediately surrounding fair-sized blood vessels in the pia and white matter of the medulla and in the posterior and lateral columns of the spinal cord. Regardless of these important lesions, the most striking parallelism between the experimentally produced and natural occurring lesions was the demyelinization, as shown by the Weigert and Weigert-Pal special stains for myelin 1 (Figs. 4 and 5). The optic nerves of the monkey that became blind were edematous and swollen (Fig. 6). In some instances, the posterior roots of the spinal nerves were found edematous and infiltrated, but in no instance were lesions found in the intervertebral ganglia. The anterior columns of the spinal cord and gray matter of the brain were conspicuously free from lesions.
Fig. 1. Area of degeneration and round cell infiltration surrounded by partially occluded blood vessels, due to endovascular and perivascular infiltration by round cells in the lateral column of the spinal cord of a rabbit, seven days after intracerebral inoculation of the streptococcus. H. and E. stain X 175.
Fig. 2. Perivascular and diffuse round cell infiltration in the wall of the lateral ventricle in the brain of a rabbit, seven days following intracerebral inoculation of the streptococcus. H. and E. stain X 175.
Fig. 3. (a) Endovascular, perivascular and diffuse round cell infiltration, degeneration and edema in the midbrain, and (b) peri-vascular infiltration, edema and degeneration in the cerebellum of a monkey, thirteen days following the first and two days following the second intracerebral inoculation of the streptococcus (Protocol 4). H. and E. stain X 175.
Fig. 4. Medulla of the rabbit, referred to in Protocol 2, twelve days following intracerebral inoculation of a 1-10,000 dilution of dextrose brain broth culture of the streptococcus. Note the unstained extensive patchy disseminated areas of demyelination especially posteriorly. Weigert myelin stain X 12.
Fig. 5. “Disseminated sclerosis—medulla. Several areas of demyelination are present in the section. These are obtained, well-defined and variable in size and shape.”7 The photomicrograph is of a person that died of multiple sclerosis. Weigert-Pal myelin stain X 9.
Fig. 6. Edema and interstitial round cell infiltration of the optic nerve of the monkey in which blindness developed (Protocol 4). Note in addition the absence of lesions within the eye and the great swelling of the optic nerve immediately outside of the inelastic sclera of the eyeball. H. and E. stain X 25.
Sections stained for bacteria revealed large or small numbers of diplococci, singly and in short chains in the lesions depending on the duration of the experiment (Fig. 8, a, b, c and d). Organisms were not demonstrable in the lesions after cultures from the brain proved sterile. Edema, infiltration by polymorphonuclear leukocytes, lymphocytes and plasma cells (Fig. 7), and the streptococcus were demonstrated microscopically in the edematous areas in the sheath and between the fibers of the optic nerve in rabbits and the monkey that had developed blurred vision or blindness (Fig 8, e). No lesions were found within the eyes in such animals (Fig. 6).
Fig. 7. Interstitial edema and round cell infiltration of the optic nerve shown in Fig. 6. H. and E. stain X 175.
Fig. 8. Diplococci or streptococci in perivascular and other areas of edema and cellular infiltration in rabbits (a, and b), and in the monkey referred to in Protocol 4: (c) in the brain, (d) in the cerebellum and (e) in the optic nerve. Modified Gram stain X 1400.
Summary and Comments
The results of a bacteriologic study of multiple sclerosis made by special methods is reported, and the mechanism by which an infective agent may cause a disease in which the usual manifestations of an infectious etiology are largely lacking is discussed. A green-producing or alpha type of streptococcus was consistently isolated from nasopharynx, tonsils and infected teeth. The cardinal symptoms of this disease were reproduced or closely simulated on appropriate intracerebral inoculation in mice, guinea pigs, rabbits and monkeys with saline suspensions of the streptococcus obtained directly from the patient, with pure cultures of the freshly isolated strains, with strains after twenty er more rapidly repeated subcultures in dextrose brain broth, and with strains after one or more passages through animals.
The “spotty” distribution of the lesions in the white matter of the brain and cord, hemorrhage, edema, demyelinization and infiltration by round cells immediately surrounding blood vessels, and other lesions in relation to vascular beds, and their similarity to the early lesions of multiple sclerosis, have been reproduced or simulated.1 Partial or complete occlusion of vessels by thrombosis, endovascular and perivascular proliferation of, or infiltration by, lymphocytes and other cells, occurred in these experiments quite as these occur in relation to the lesions of multiple sclerosis,2,8 and the lesions of other diseases of the nervous system.18
Lesions of the lungs in rabbits and mice developed not infrequently following inoculation of the streptococcus, which is in accord with the fact that the onset of the disease and especially exacerbations or extensions often follow attacks of influenza or other respiratory infections.8,17
The different strains isolated during the active stage of the disease were agglutinated specifically by the serums of persons stricken, by antiserums prepared with closely related streptococci, such as those from encephalitis, and by thermal antibody prepared in vitro from streptococci isolated in studies of multiple sclerosis.
Specific streptococcic antigen was demonstrated in skin or blood of persons in the active stage of the disease by intradermal injection of solutions of the respective closely related “natural” and specific streptococcal thermal antibodies, and specific streptococcal antibody was demonstrated by intradermal injection of streptococcal antigen. Cutaneous reactions indicating antigen were greatest during the active stage of the disease, became less marked as active symptoms subsided and as antibody increased, and both became slight or absent during the quiescent stage.
Intravenous therapeutic injections of histamine, and especially of histamine and thermal antibody as given under Dr. Horton’s supervision,3 and the thermal antibody and antigen, or vaccine without histamine, caused a diminution of antigen and an increase in antibody and apparently a concomitant improvement in symptoms in persons during the active stage of multiple sclerosis. A nonspecific and specific means for treatment seem at hand. Due regard to the prevention of respiratory infection and to a consideration of foci of infection, especially in teeth and tonsils, is indicated.
The cause of death in animals in which cultures of the brain revealed the streptococcus seemed clear, but in those that died after the streptococcus was no longer isolable from the brain or blood, and no longer demonstrable in the lesions simulating in this respect what occurs in multiple sclerosis, the cause of death was obscure. Attempts were made to explain this phenomenon. The evidence adduced indicates that fatalities in the experimental and naturally occurring disease, in the absence of living streptococci, may be due to the formation of a streptococci neurotoxin having predilection for vital nerve centers, and to which vital centers become allergic, and perhaps to the formation of an autogenous sensitizing streptococcal-nerve-tissue complex which may function in a manner similar to the wholly foreign adjuvant-nerve-tissue complexes used successfully by others in the production of “allergic” encephalomyelitis.4,5
The possibility of a virus etiology has not been sufficiently studied. The data obtained indicate that a green-producing or alpha streptococcus of low general virulence, having specific localizing, toxicogenic and antigenic properties, is etiologic in multiple sclerosis.
References Cited:
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- Dawson, J. W.: The histology of disseminated sclerosis. Roy. Soc., Edinburgh, 50:517, 1916.
- Horton, B. T.: Personal communication; and A.M.A., 124:800-801, 1944.
- Kabot, E. A.: Wolf, A., and Bezer, A. E.: Rapid production of acute disseminated encephalomyelitis in rhesus monkeys by injection of brain tissue with adjuvants. Science, 104:362-363, 1940.
- Morgan, T. M.: Allergic encephalomyelitis in monkeys in response to injections of normal monkey cord (with adjuvant). Johns Hopkins University, Poliomyelitis Research Center, Baltimore, Maryland.
- Noran, H. H.: Baker, A. B., and Larson, W. P.: Central nervous system in pneumonia (non-suppurative pneumonic encephalitis). Neural & Psychiat., 58:653-671, 1947.
- Ogilvie, R. F.: Pathological Histology, Baltimore: Williams & Wilkins Co., 1943.
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- Rosenow, E. C.: Studies on focal infection, elective localization arid cataphoretic velocity of streptococci: Dental Cosmos, 76:721-744, 1934.
- Rosenow, E. C.: Demonstration of the association of specifically different alpha streptococci with various diseases and methods for the preparation and use of specific antiserums and, vaccines in diagnosis and treatment. J. Clin. Path., 12.339-356, 1942.
- Rosenow, E. C.: The production in vitro of substances resembling antibodies from bacteria. Infect. Dis., 76:163-178, 1945.
- Rosenow, E. C.: Studies on the relation of pneumotropic streptococci to influenza virus. J. Clin. Path., 15:362-380, 1945.
- Rosenow, E. C.: Studies on the nature of antibodies produced in vitro from bacteria with hydrogen peroxide and heat. Immunol., 55:219-232, 1947.
- Rosenow, E. C.: Diagnostic cutaneous reactions to intradermal injection of natural and artificial antibody and of antigen prepared from streptococci isolated in studies of diverse diseases. Allergy, (in press).
- Rosenow, E. C.: Bacteriologic, etiologic and serologic studies in epilepsy and schizophrenia, II. Med., 3:124-136, 1948.
- Rosenow, E. C., and Nickel, A. C.: Results in various diseases from elimination of foci of infection and use of vaccines prepare from streptococci having elective localizing power. Lab. & Clin. Med., 14:504-512, 1929.
- Scheinker, I. M.: Vaso Paralysis and vaso thrombosis of the brain in infancy and in early childhood. Neural. & Psychiat., 55:216, 1946.