STUDIES OF THE VARIATIONS IN THE ANTISTREPTOLYSIN TITER OF THEBLOODSERUMFROMPATIENTS WITH HEMORRHAGICNEPHRITIS. I.

CONTROLOBSERVATIONSONHEALTHYINDIVIDUALS ANDPATIENTS SUFFERINGFROMDISEASES OTHER

THAN STREPTOCOCCALINFECTIONS

By WARFIELDT. LONGCOPEI

(From the Medical Clinic, the School of Medicine, Johns Hopkins University and Hospital,Baltimore)

(Received for publication January 15, 1936)

During some studies which have been in progress forseveral years upon the etiology and the clinical course ofacute glomerular nephritis, it was considered desirable toexamine the serum from these patients for antibodies tohemolytic streptococci. Comment has frequently beenmade to the effect that infections by this organism in-volving the upper respiratory tract usually precede theonset of acute hemorrhagic nephritis, and particular at-tention has been drawn to this matter in previous publi-cations (1). Efforts to demonstrate the presence ofspecific agglutinins and specific precipitins for hemolyticstreptococci in the serum from cases of acute, subacuteand chronic glomerular nephritis were met with indiffer-ent success and very irregular results, and the investiga-tions were being abandoned when Todd published his ob-servations on antistreptolysin.

Todd (2) showed that the hemolysin produced byhemolytic streptococci, grown in a special medium freeof animal sera, was antigenic and gave rise in rabbits toan antistreptolysin of considerable potency. Streptolysinformed by the growth of hemolytic streptococci in serumbroth did not excite antibodies capable of neutralizingserum broth streptolysin nor would antistreptolysin ofhigh titer neutralize serum broth streptolysin. Since itseemed probable that antistreptolysin might be formed inthe serum of animals immunized to hemolytic strepto-cocci, Todd tested scarlatinal antitoxin, erysipelas anti-toxin and puerperal septicemia antitoxin, and found thatantistreptolysin was present in high titer. Whereas theminimal neutralizing dose of normal horse serum was 0.2cc. to 0.04 cc., the minimal neutralizing dose of the spe-cific antitoxins was from 0.005 to 0.0002 cc. He furtherobserved that the titers of diphtheria antitoxin, tetanusantitoxin, staphylococcus antitoxin, Small's non-hemo-lytic streptococcal antitoxin, antipneumococcal serum,antimeningococcal serum, and anti-gas gangrene serumdid not differ from those of normal horse serum. Todd(3) then studied the reaction of the blood serum of manduring infections by hemolytic streptococci. He recordedthe antistreptolysin values of serum in units which arecalculated as the reciprocal of the fractions of the cubiccentimeters of serum employed. Under these conditions0.01 cc. of serum equals 100 units, 0.03 cc. is equivalent

1 The titrations of antihemolysin were made with thetechnical assistance of Cyril Skala.

to 33.3 units and so forth. He found that the titers ofsera from normal individuals varied between 20 unitsand 100 units. During convalescence from scarlet fever,erysipelas and tonsillitis, the antistreptolysin of the serumwas elevated and sometimes reached 1,000 to 1,300 units.The serum from a limited number of patients sufferingfrom infections that were not associated with hemolyticstreptococcal infections gave titers of approximately nor-mal values. During the acute phase of severe or fatalinfections by hemolytic streptococcus the antihemolytictiters of the serum were not elevated. In some observa-tions made upon the serum from nurses at the Presbyte-rian Hospital in New York, Todd found that during theperiod of normal health, or when persons were simply ex-posed to hemolytic streptococcal infections, that the anti-streptolysin content of the serum was within normal lim-its. During the acute phase of streptococcic pharyngitisthe titers still remained low, but during convalescence thetiters rose and remained high for as long as one to threemonths after the infection. Todd extended his observa-tions to rheumatic fever and found that the sera of pa-tients in the quiescent stage gave titers that were withinnormal limits. During the active stage, however, theantistreptolysin content of the serum was much increased.This increase of antistreptolysin appeared to be dependentupon the preceding streptococcal infection of the respira-tory tract, and did not vary with the severity of the at-tack of rheumatic fever. The antistreptolysin values ofthe serum often remained high for months during the at-tack of rheumatic fever and into the period of conva-lescence. Todd concluded that his observations affordedevidence that rheumatic fever was preceded by hemolyticstreptococcal infections.

These observations on rheumatic fever were immedi-ately confirmed and extended by Coburn and Pauli (4).They found that in the normal individual the neutralizingdose of serum was usually 0.02 cc. but might be as smallas 0.01 cc. or 100 units, but rarely less. Following anattack of scarlet fever or erysipelas the titer rose from anormal figure to 0.003 or even as high as 0.0008 cc. in acase of erysipelas. In the acute rheumatic process theneutralizing dose of serum often reached 0.005 cc. orless. In inactive rheumatic fever the titers of the serawere usually within normal limits. The antistreptolysintiter of the serum remained normal during the infectious

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WARFIELD T. LONGCOPE

and quiescent period preceding the acute attack of rheu-matic fever, but rose abruptly just before the appearanceof rheumatic symptoms. During the first week of rheu-matic fever it reached its highest level, and maintainedthis throughout the attack. They believed that their ob-servations furnished strong evidence that the rheumaticattack was initiated by hemolytic streptococci. Since thisfirst publication, Coburn and Pauli (5) have extendedtheir studies and reference will be made to their resultslater on.

Since the appearance of the first papers by Todd andby Coburn and Pauli, a considerable number of articleshave been published on the subject. Griffiths (6) hasfound high antistreptolysin in titers in all cases of acuterheumatic fever, and in a much smaller proportion of avariety of arthritic conditions. He considered 50 unitsas the normal. Myers and Keefer (7) give rather highfigures for the normal, but since they modified the methodof Todd in preparing streptolysin, and employed sheepred blood cells instead of the red blood cells from rabbitstheir figures can scarcely be compared with those pub-lished by others. They confirmed the preceding observa-tions by finding high titers in proven hemolytic strepto-coccal infections, and comparatively high titers in acuterheumatic fever. They did not find an increase in theantistreptolysin content of the serum from patients withrheumatoid arthritis or other joint infections. Theycould not relate the antistreptolysin content of the serumto agglutinins for hemolytic streptococci or to the skinsensitivity to the nucleoprotein of the Streptococcws scar-latinae. The studies of Wilson, Wheeler and Leask (8)did not support the assumption that a rise in the anti-streptolysin titer of the serum was conclusive evidence ofstreptococcal respiratory infection, and they stated thata rise in the antistreptolysin titer was not a necessary ac-companiment of rheumatic fever in children.

Lippard and Johnson (9) have shown, however, thatthe antistreptolysin content of the serum of normal in-fants under 17 months of age is low, the average being24 units and 55 per cent being 2 units or less. The av-erage for children over 17 months of age was 66 units.In the infants there was little tendency for the antistrep-tolysin titer of the serum to rise following infections byhemolytic streptococci, whereas in older children theresponse was comparable to that observed in adults. Itwas further found (10) that the treatment of hemolyticstreptococcal infections of infants and children by trans-fusions of blood or injections of streptococcal antiserawas not accompanied by a significant increase in anti-streptolysin substance in the blood of the recipient. Blairand Hallman (11) titrated antistreptolysin in the serafrom a few normal individuals and from patients suffer-ing from a variety of conditions. They concluded that100 units is the upper limit of normal. The antistrepto-lysin titer was moderately increased in about one-thirdof the sera from 45 cases of rheumatoid arthritis, veryhigh in the sera from 7 cases of streptococcal infectionas well as in 15 out of 18 cases of acute rheumatic fever.They concluded that a high titer of antistreptolysin in

blood serum signifies specifically infection by hemolyticstreptococci.

Owing to the fact that many observers consider thatthe presence of distinctly abnormal amounts of antistrep-tolysin (over 200 units) in the blood serum is a definiteindication of recent or existing infection by hemolyticstreptococci, the test has been employed by some to detectsuch an infection. Morales-Otero and Pomales-Lebron(12) tested the antistreptolysin titer of the sera from 41cases of recurrent lymphangitis in Porto Rico, and sincethey often found the titers elevated considerably abovethat of their normal controls, they suggest that this con-dition is due to a recurring infection by hemolytic strep-tococci. Seegal and Lyttle (13) have determined theantistreptolysin titers of the serum from 22 cases of acuteglomerular nephritis. An infection of the throat or up-per respiratory tract preceded the onset of nephritis in21 of these cases and in 15 of them the infections wereassociated with hemolytic streptococci. The sera wereexamined 6 to 57 days after the onset of the acute nephri-tis. Twenty of the 22 sera showed an increase of anti-streptolysin titers ranging from 125 to 1250 units. Theyconsidered that their data offered strong evidence to sup-port the contention that acute glomerular nephritis ischiefly related to a preceding hemolytic streptococcalinfection.

It is unnecessary to repeat in detail the methodof making the medium and preparing streptolysin.The directions given by Swift and Hodge (14),who were kind enough to acquaint us with thetechnique before their publications, were followedprecisely. Nor is it desirable to repeat the detailsof performing the test, for this has been accu-rately described by Coburn and Pauli (5), andtheir method has been strictly adhered to. Todd'soriginal "Aronson" strain of hemolytic strepto-coccus, which was kindly sent to us by Drs. Swiftand Hodge, has been the organism used. Com-plete hemolysis has usually been obtained withfrom 0.1 cc. to 0.08 cc. of streptolysin, after ithad remained sealed under vaseline at 40 C. for2 to 3 weeks. The hemolytic titer of each batchof hemolysin preserved in separate tubes has re-mained practically unchanged for months. Afresh tube of streptolysin has been used for eachseries of tests. Even though Hodge and Swift(15) have pointed out that the combining unit ofstreptolysin with serum may remain constantwhen the hemolytic dose varies somewhat, thehemolytic dose of each tube of streptolysin hasbeen determined before the tests with serum weremade. As a rule 2% minimal hemolytic doses

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VARIATIONS IN ANTISTREPTOLYSIN TITER OF BLOODSERUM. I

proved to be the amount which gave a constantcombining power. A 5 per cent suspension offreshly drawn rabbits' red blood cells washed 6times in 0.85 per cent NaCl was used for thetests. Each batch of streptolysin was titeredcarefully against a serum of known antistrepto-lysin content before it was employed for the tests.The first lots of tested serum were sent to us byDrs. Swift and Hodge. Later, Dr. Todd verykindly sent us immune serum, the titer of whichwas 20,000 units per cc. In each series of tests aserum of known low titer and a serum of knownhigh titer has been used for control. For manymonths the serum sent by Dr. Todd was the oneemployed for the high titer control. The testshave been read shortly after removal from thewater bath and again after standing for 18 hoursat 4° C. Except in the rarest instances the read-ings coincided. When they did not coincide thetest was repeated.

It seemed obvious that if a study of the anti-streptolysin content of the serum from patientswith hemorrhagic Bright's disease was to be ofany value, one should in the first place examinesamples of serum from the same patient at fairlyfrequent intervals, and in the second( place maketests on the serum from a number of normalindividuals and from patients suffering from avariety of diseases in order that a fair backgroundfor comparisons might be obtained.

To this end we have examined more than 1,716samples of sera for the antistreptolysin contentfrom 55 supposedly normal individuals and from516 patients suffering from a variety of condi-tions. The observations, for clarity of discus-sion, can best be divided into two parts. Thefirst part deals with the results obtained from thetitration for antistreptolysin in the serum from(a) presumably normal individuals, (b) from pa-tients suffering from such chronic diseases asdiabetes mellitus and exophthalmic goiter, (c)from patients affected by various forms of ane-mia, leukemia and new growths, (d) from pa-tients with chronic inactive rheumatic heart dis-ease, (e) from patients with essential hyper-tension, hypertensive heart disease, chronic vas-cular nephritis and syphilitic aortitis, and finallyfrom patients suffering from a variety of infec-tious diseases not associated with hemolytic strep-tococci.

In order to obtain a fair average of the naturalantistreptolysin content of the serum, 84 samplesfrom 55 supposedly normal individuals have beenexamined. The results are given in Table I. Ineach instance the highest titer is recorded. In40 individuals, or in 74.5 per cent, the titer lay

TABLE I

Antistreptolysin titers of the blood serum from 55 normalindividuals and 84 patients suffering from

chronic disease

Antistreptolysin titers (unite per cc.)

Diagnosis

.qr Po cqc --

Normal ............. 3 5 1 20 1 19 5 1 55 1Hyperthyroid and

myxedema 1 2 4 7 0Diabetes mellitus .1.. 1 2 1 4 1 9 1Anemias, chronic leu-

kemia paroxysmalhemo lobinuria,

of lung, infectiousmononuleosis ...... 2 4 7 2 15 2

Chronic rheumaticheart disease 6 5 2 3 2 18 5

Hypertension, chronicvascular nephritis,syphilitic aortitis,arteriosclerotic heartdisease............ 1 111114 1 5 1 35 6

Total..... .1 4 11 44 538 9_ 5 139 15

between 25 and 50 units; in 3 it was 12.5 units,and in 5 it was 100 units. In only one personwas it above 100 units. In this one individual,who was apparently normal, repeated culturesfrom the throat failed to show either beta hemo-lytic streptococci or the minute hemolytic strepto-cocci described by Long and Bliss (16); the titeron two occasions over a period of 3 months was142.9. Eight months after the last titration, theserum showed a titer of 100. These results aresimilar though perhaps somewhat more uniformthan those obtained by Coburn and Pauli (5, Iand VI) from a much larger series of individualsin New York. Seventy-five per cent of 146 per-sons showed titers of 100 or below, and the me-dian titer for 176 persons in good health was 83units.

Though the usual titer for antistreptolysin ap-peared to fall between 25 and 50 units for theadults in our series, a result that corresponds tothe natural titer (5, I), it seemed desirable to de-termine whether variations might occur in a singleindividual's serum over a fairly long period dur-

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WARFIELD T. LONGCOPE

ing which time he would probably contract severalminor infections. The sera from five normal in-dividuals and from one case of hypertension withchronic nephritis have been examined at intervalsover periods varying from 7 months to one year.The results are given in Table II. It may beseen that the titers remain comparatively constantin spite of colds and attacks of bronchitis and,with the exception of Number 3 already referredto, they fall well within the limits of the naturalantistreptolysin for the larger group.

TABLE II

Antistreptolysin titers (units per cc.) of the blood serumfrom 6 individuals over a period of 7 to 13 months

Timeof Patient Patient Patient Patient Patient Patient

determi- it 2t 3t 4t 5t 6tnation

months1 50.0 33.3 142.9 50.0 16.7 25

50.0 16.72 50.0 50.0 25

50.0 50.03 50.0 142.9 100 204 50.0 Severe

50.0 cold5 50.0 33.3 50.0 206 50.0 33.3

50.07 50.0 50.0 16.7

33.389 50.0

50.050.0

10 50.050.050.050.0

11 10012 50.0 100 33.3* 20

125 2013 12514 100

t Comments: Patient 1. Normal. Occasional colds.2. Normal. 3. Normal. Chronic pharyngitis-repeatedcultures show no B. hemolytic streptococcus or minuteB. hemolytic streptococcus. 4. Normal. Several severecolds; infection of finger at * with B. hemolytic strepto-coccus. 5. Normal. Repeated severe colds. 6. Hyper-tension with chronic vascular nephritis.

The determinations of the antistreptolysin con-tent of sera from all the patients suffering fromthe various chronic diseases may be consideredtogether. The results are compared in Table Iwith the normal individuals. In each instance

the highest titer obtained is recorded. One hun-dred and fifty specimens of serum were exam-ined from 84 patients. In several instances fiveor more collections of serum were made over aperiod of several weeks or months. The titersremained remarkably constant for each individ-ual. In general the titers vary little from thenatural antistreptolysin content of serum. In 50patients, or a little less than 60 per cent, the titersfell between 25 and 50 units; in one case of dia-betes the titer was very low for the normal adult-10 units. This corresponds to the very lowtiters found by Lippard and Johnson (9) in in-fants under 18 months of age. In 14 patients, or16.6 per cent, the titers were slightly elevatedabove the natural content. There was no obviousexplanation for this increase.

The infectious diseases that are included in Ta-ble III were typhoid fever, malaria, bacterial en-docarditis, meningococcal meningitis and gono-coccal arthritis, a miscellaneous group includingtuberculosis, epidemic jaundice, bronchitis, peri-arteritis nodosa and vesicular stomatitis, andfinally lobar pneumonia. There were 66 indi-viduals in this group.

TABLE III

Antistreptolysin titers of the blood sera from 66 patientssuffering from various forms of acute infection

Aintisteptobain titer (units per cc.) S

Diagnodsis- _ _ _

TyphoId . ..... ......212 11 1 6 11 2Malaria . ~2 1 3 1 7 8 1

Bacterial endocardi-tis 5 1 1 1 8 21 2

Meningococcusmen-gitis,gonococcus

arthritis ........ 1 1 3 1 2 8 9 3Miscellaneous, in-

cluding tubercu-losis, bronchitispyelitis 2 8 2 1 13 20 3Pneumona 8192 1 3 24 44 4

Total...... 3 13 130 4 14 8 2 66 113 15

One hundredwere examined,

and thirteen specimens of serumand frequently several bleedings

were made at intervals from the same patient.Table III gives the results of these examinations.In each instance the highest antistreptolysin titerobtained was recorded.

It will be seen that though a slightly larger

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VARIATIONS IN ANTISTREPTOLYSIN TITER OF BLOOD SERUM. I

proportion of these patients (22.7 per cent) thanthose suffering from chronic diseases (16.6 percent) gave titers of antistreptolysin above thenormal level, only two patients, one an instance ofbacterial endocarditis and one a patient with pyeli-tis, showed an increase up to 250 units, a levelwhich most observers consider to be highly sig-nificant. Since it has been remarked repeatedlythat the antistreptolysin titer of the serum in in-fections due to hemolytic streptococci does notincrease until the disease is well advanced or untilconvalescence has been well established (3, 4, 5),it is important to know the day of disease onwhich the tests were made. The serum fromthree of the patients with typhoid fever was testedbetween the third and sixth week of the disease,and from three patients between the seventh andeleventh week of the disease. In one of thesepatients the serum was tested at weekly intervalsfor three weeks. The titer remained constantlyat 50 units. In one, the serum was tested weeklyfor four weeks. The titer in the eighth week was100 units; in the ninth, tenth and eleventh weeksit remained at a constant titer of 111.1 units.The sera from the cases of malaria, of meningo-coccal meningitis and of gonococcal arthritis weretitrated only during the acute phase of the disease.The sera from the miscellaneous cases were testedat various stages of their diseases, and the serafrom the cases of bacterial endocarditis weeks andmonths after the onset of their illness.

Especial interest attaches to the examination ofthe patients with lobar pneumonia, for Todd (17)has reported the fact that antistreptolysin serumof high titer will neutralize the oxygen labilehemolysin produced by pneumococci and by B. te-tanus. The reaction was found to occur, how-ever, only with "hyperimmune serum." Thispartial overlapping was not noticed when theserum from patients suffering from hemolyticstreptococcal infections, which is of low titercompared with the serum from immunized ani-mals, was studied. The serum from nine pa-tients convalescing from lobar pneumonia due topneumococcus Type II were tested for antistrep-tolysin. The serum from only one patient gavean abnormally high figure of 250 units.

The sera from 24 cases of lobar pneumonia,which are listed in Table III, were collected from

the third to the fifth week of the disease. Thesera from 20 patients gave titers on repeated ex-amination that were quite constant and alwayswithin normal limits. The sera from 4 patientsgave titers that were equally constant but slightlyabove normal. The titrations were made in thesecond, third and fourth week of the disease.The titers varied from 125 units to 166.7 units.The pneumonia in three of these patients was dueto pneumococcus Group IV and in one to pneu-mococcus Type I. The blood cultures were nega-tive in all four cases and all recovered. Of theremaining 20 cases, 5 gave positive blood cul-tures; two of these died, three recovered. Threepatients infected with pneumococcus Type I re-ceived concentrated antipneumococcal serum andrecovered. Of the 24 cases, 14 were infectionsdue to pneumococcus Type IV; 7 were due topneumococcus Type I; one to pneumococcus TypeII; one to pneumococcus Type III, and in onethe organism was not identified.

The proportion of slightly elevated titers was,therefore, not quite as large in these patients suf-fering from pneumonia as it was in the meningo-coccal and gonocccal infections.

SUMMARY

In reviewing the results obtained from titra-tions for antistreptolysin in the sera from normalindividuals, and from patients suffering from avariety of chronic diseases and acute infectionsnot caused by hemolytic streptococci, it is seenthat they accord in general with those previouslypublished. It appears justifiable to conclude, asTodd did originally, that the upper limit of anti-streptolysin in the serum from the normal adultis 100 units. In the majority of cases (about 75per cent) the titer in our series lay between 25and 50 units. The determination of the lowerlimit of the antistreptolysin in the normal adultis also important, for Lippard and Johnson (9)found this to be unusually low in infants under17 months of age. In 55 per cent of these pa-tients the titer was only 2 units. The lower levelin our normal adults was 12.5 units. It is alsoto be observed that the titer varies very little frommonth to month in a given individual. The titer.appeared to be unaffected by severe colds and mi-nor infections not due to hemolytic streptococci,

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WARFIELD T. LONGCOPE

but even a comparatively insignificant infectioncaused by hemolytic streptococci may result, as itdid in Number 4, in a significant rise in the anti-streptolysin titer within 18 days of the infection.Since such rises, as has been repeatedly shown,may persist for weeks or months, it would not besurprising to find a moderately elevated antistrep-tolysin titer in the serum of individuals who were

apparently healthy, who showed no evidence ofinfection by hemolytic streptococci and who were

actually unaware of having had such infections.In view of this possibility it is noteworthy that

the antistreptolysin content of the sera from pa-

tients suffering from chronic diseases and from a

miscellany of infections should approach so

closely the normal figure. In the examination of155 specimens of serum from the 84 patientswith chronic disease only a total of 16 samplesshowed titers above 100 units and none of thesewas above 166.7 units. The total number ofpatients furnishing serum showing titers above100 units was 14, or 16.6 per cent of the total.

Of the 155 samples of serum from the 84 pa-

tients, 139 gave titers within the average limitsof normal.

In the acute infections the number of slightlyelevated titers was proportionately greater, forof the 113 samples of serum from 66 patients,22 samples from 15 patients gave antistreptolysintiters somewhat above the figure for normaladults. Titers above 100 units occurred in 22.7per cent of the 66 patients.

It would be impossible to determine, at thepresent tithe, whether the infecting organism, suchas the meningococcus or pneumococcus, was inany way related to these abnormal rises, or

whether an infection by hemolytic streptococcicontracted previous to, or in association with thepresent infection was responsible for the abnor-mal titer. Owing to the prevalence of hemolyticstreptococci it would be impossible to exclude thelatter possibility.

BIBLIOGRAPHY

1. Longcope, W. T., O'Brien, D. P., McGuire, J., Han-sen, 0. C., and Denny, E. R., Relationship of acuteinfections to glomerular nephritis. J. Clin. In-vest., 1927, 5, 7.

Longcope, W. T., The pathogenesis of glomerularnephritis. Bull. Johns Hopkins Hosp., 1929, 45,335.

Lukens, F. D. W., and Longcope, W. T., Experi-mental acute glomerulitis. J. Exper. Med., 1931,53, 511.

Longcope, W. T., Bordley, J., III, and Lukens, F. D.W., The Kidney in Health and Disease. ChapterXXI. Infection by Streptococci in Relation toRecovery and Progress of Nephritis. Lea andFebiger, Philadelphia, 1935.

2. Todd, E. W., Antigenic streptococcal hemolysin. J.Exper. Med., 1932, 55, 267.

3. Todd, E. W., Antihaemolysin titres in haemolyticstreptococcal infections and their significance inrheumatic fever. Brit. J. Exper. Path., 1932, 13,248.

4. Coburn, A. F., and Pauli, Ruth H., Studies on therelationship of streptococcus hemolyticus to therheumatic process. III. Observations on the im-munological responses of rheumatic subjects tohemolytic streptococcus. J. Exper. Med., 1932, 56,651.

5. Coburn, A. F., and Pauli, Ruth H., Studies on theimmune response of the rheumatic subject and itsrelationship to activity of the rheumatic process.I. The determination of antistreptolysin titer. J.

Exper. Med., 1935, 62, 129.II. Observations on an epidemic of influenza fol-

lowed by hemolytic streptococcus infectionsin a rheumatic colony. Idem, p. 137.

III. Observations on the reactions of a rheumaticgroup to an epidemic infection with hemo-lytic streptococcus of a single type. Idem,p. 159.

IV. Characteristics of strains of hemolytic strepto-coccus, effective and non-effective in initiat-ing rheumatic activity. J. Clin. Invest., 1935,14, 755.

V. Active and passive immunization to hemolyticstreptococcus in relation to the rheumaticprocess. Idem, p. 763.

VI. The significance of the rise of antistreptolysinlevel in the development of rheumatic ac-tivity. Idem, p. 769.

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7. Myers, W. K., and Keefer, C. S., Antistreptolysincontent of the blood serum in rheumatic fever andrheumatoid arthritis. J. Clin. Invest., 1934, 13,155.

8. Wilson, May G., Wheeler, G. W., and Leask, M. M.,Antistreptolysin content of blood serum of chil-dren. Its significance in rheumatic fever. Proc.Soc. Exper. Biol. and Med., 1934, 31, 1001.

The relation of upper respiratory infections to rheu-matic fever in children. II. Antihemolysin titresin respiratory infections and their significance inrheumatic fever in children. J. Clin. Invest., 1935,14, 333.

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VARIJTIONS IN ANTISTREPTOLYSIN TITER OF BLOODSERUM. I

9. Lippard, V. W., and Johnson, P., Beta hemolyticstreptococcic infection in infancy and in childhood.I. Antifibrinolysin and antistreptolysin response.Am. J. Dis. Child, 1935, 49, 1411.

10. Ibid. II. Effect of transfused blood and of strepto-coccic antiserum on the concentrations of antifi-brinolysin and antistreptolysin in the blood of therecipients. Am. J. Dis. Child., 1935, 49, 1430.

11. Blair, J. E., and Hallman, F. A., Streptococcalagglutinins and antistreptolysins in rheumatoid(atrophic) arthritis. J. Clin. Invest., 1935, 14, 505.

12. Morales-Otero, P., and Pomales-Lebron, A., Anti-streptolysin content of sera from cases of recur-rent tropical lymphangitis. Proc. Soc. Exper.Biol. and Med., 1934, 31, 1170.

13. Seegal, D., and Lyttle, John D., Antistreptolysin titreof the serum in acute glomerular nephritis. Proc.Soc. Exper. Biol. and Med., 1933, 31, 211.

Seegal, B. C., Seegal, D., Jost, E. L., and Cameron,P., Bacteriological and immunological studies ofacute glomerulo-nephritis in New Orleans. Proc.Soc. Exper. Biol. and Med., 1935, 32, 1151.

14. Swift, H. F., and Hodge, B. E., A simply preparedbroth for producing hemolytic streptococcal hema-toxin (streptolysin), Proc. Soc. Exper. Biol. andMed., 1933, 30, 1022.

15. Hodge, B. E., and Swift, H. F., Varying hemolyticand constant combining capacity of streptolysins;influence on testing for antistreptolysins. J. Ex-per. Med., 1933, 58, 277.

16. Long, P. H., and Bliss, E. A., Studies upon minutehemolytic streptococci. I. The isolation and cul-tural characteristics of minute beta hemolyticstreptococci. J. Exper. Med., 1934, 60, 619.

Long, P. H., Bliss, E. A., and Walcott, C. F., II.The distribution of minute hemolytic streptococciin normal and diseased human beings. J. Exper.Med., 1934, 60, 633.

17. Todd, E. W., A comparative serological study ofstreptolysins derived from human and from animalinfections, with notes on pneumococcal hemolysin,tetanolysin and staphylococcus toxin. J. Path. andBact., 1934, 39, 299.

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