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STUDIES OF GONOCOCCALINFECTION. I. A STUDYOF THEMODEOFDESTRUCTIONOFTHEGONOCOCCUSIN VITRO'

By WESLEYW. SPINK AND CHESTERS. KEEFER(From the Thorndike Memorial Laboratory, Second and Fourth Medical SerAces (Harvard),

Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston)

(Received for publication October 7, 1936)

At the present time, little is known concerningthe development of local or general immunity inpatients with gonococcal infections. It is uni-versally recognized that local infections are no-toriously chronic and that repeated infections ofthe same individual are common. The study ofthe immune mechanism in animals has not beenvery satisfactory since it is an extremely difficultmatter to establish a gonococcal infection in ordi-nary laboratory animals. It did not seem un-reasonable to suppose that more information couldbe obtained concerning the mechanism of immu-nity from a study of several serological reactionsthat appear during the course of the natural in-fection in man. We have studied, therefore,various antibodies in the blood of patients withdifferent types of gonococcal infection, viz., thecomplement-fixing antibodies, agglutinins, precipi-tins, -and the bactericidal power of defibrinatedwhole blood upon the gonococcus. We havefound a study of the bactericidal power of wholeblood most serviceable, and at this time we pre-sent a detailed discussion of its action, togetherwith an analysis of the action of the circulatingleukocytes and serum, upon the gonococcus. Theresults of this and the other tests upon a series ofpatients with gonococcal infections and normalcontrols are presented in the second paper.

METHODS

The bactericidal action of whole defibrinatedblood, serum, or plasma was determined by themethod of Todd as used by us (1) in studyingthe hemolytic streptococcus. To 0.5 cc. of wholedefibrinated blood, serum, or plasma, in smallpyrex tubes, varying dilutions of an 18-hoursuspension of gonococci in ascitic fluid broth wereadded. The number of organisms in each tubewas determined by the plating of 1 cc. of the con-

' This investigation was aided, in part, by a grant fromthe Milton Fund and Clark Bequest.

tents of tubes 10-6 and 10-i. The tubes weresealed in a gas-oxygen flame and then rotated, ina box, for 36 to 48 hours in an incubator at 37.50C. The tubes were then opened and the contentswere cultured to determine the presence of livingorganisms.

Thirty-six strains of gonococci isolated fromactive cases of gonorrhea have been studied in thismanner. They were obtained from urethral andcervical exudates, synovial fluid, tendon sheaths,conjunctivae, and the circulating blood. Thestrains were grown on horse blood agar plateswith a pH of 7.8 and sub-cultured every 3 days.The identity of the organisms as gonococci wasproved by agglutination reactions with a poly-valent antigonococcal serum, and by sugar fer-mentation tests.

The action of the leukocytes upon the gono-coccus was investigated in two ways. First,quantitative phagocytic studies were made on theblood of patients with gonococcal arthritis, andof normal controls. The procedure was to add0.1 cc. of an 18-hour ascitic fluid broth culture ofgonococci to 0.5 cc. of whole defibrinated bloodin small pyrex tubes. After sealing the tubes,they were rotated in the incubator for 15 minutesto 4 hours. It was found that 15 minutes wasthe optimal time for maximal phagocytosis to takeplace. Smears were made and stained withWright's stain. Fifty polymorphonuclear leuko-cytes were counted, and the number of cells con-taining organisms was noted. The degree ofphagocytosis was compared with the bactericidalaction of whole blood on the same suspension oforganisms. The second method was to removethe plasma from whole defibrinated blood andwash the cells 4 times with 0.85 per cent salt solu-tion. One-tenth of a cubic centimeter of a sus-pension of gonococci was added to 0.25 cc. ofcells suspended in 0.25 cc. of salt solution in pyrextubes. The tubes were sealed and rotated for15 minutes, and then opened to determine the

169

WESLEYW. SPINK AND CHESTERS. KEEFER

presence of living organisms and the degree ofphagocytosis.

RESULTS

Bactericidal action of whole defibrinated blood,serum and plasma

The results of 33 observations on the bacteri-cidal action of the whole defibrinated blood andof serum or plasma alone are presented in Table I.Thirteen different strains of gonococci were used.One specimen of blood was obtained from each of13 patients with various types of gonococcal le-sions and 2 normal controls. It is to be notedthat equivalent amounts of serum had essentiallythe same killing power as whole, defibrinatedblood. Tests were also done with plasma to de-termine whether it had the same killing power as

serum or whole blood. An examination of

Wright's stained smears made from the blood inwhich there was growth of organisms revealedthat most of the polymorphonuclear leukocytescontained gonococci. However, similar smears ex-

amined from the blood in the tubes in which therewas no growth failed to show either extracellularor intracellular organisms. In short, it wouldappear that when the gonococcus was killed bythe blood, it underwent complete lysis.

Bactericidal action of whole defibrinated blood,plasma and heated plasma

It has been indicated already that the killingpower of whole defibrinated blood for the gono-

coccus is essentially the same as that of plasmaor serum. If this bacteriolytic action on thegonococcus is due to the combined action of anti-

ILE I

Comparison of bactericidal power of whole defibrinated blood, serum, and plasma *(Each row represents a tenfold dilution of the gonococcus)

Obser- Sthin Whole defibrinated blood Serum Plasma Numbervation Patient num- ---------- ofnu.- numbernu-_ ._ _ o

bu n ber 10-1 10' 10' 10-4 106 10-6 10' 101 105 l)' 10-4 10- 10-6 10) 101- 105 103 10-4 10-' 10-' 10 organisms

1 1 1 + + 0 0 0 0 0 + + + 0 0 0 0 10-7=52 2 2 + + 0 0 0 0 0 + + 0 0 0 00100 '=63 3 3 + 0 0 0 0 0 0 0 0 0 0 0 0 0 10-=54 4 4 + + 0 0 0 0 0 0 0 0 0 0 °0 10-7==95 5 5 + + + 0 0 0 0 + + + 0 0 0 0 10-7=86 6 6 + 0 0 0 0 0 0 0 0 0 0 0 0 0 10-'=37 6 + 0 0 0 0 0 0 0 0 0 0 0 0 0 10-'=38 13 + 0 0 0 0 0 0 0 0 0 0 0 0 0 10-'=49 7 7 + 0 0 0 0 0 0 + 0 0 0 0 0 0 10-4=20

10 8 + 0 0 0 0 0 0 + 0 0 0 0 0 0 10-'=411 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 712 8 8 + + + 0 0 0 0 + + + 0 0 0 0 10-=413 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10'=314 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10-7= 1215 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 106=316 11 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 I-'=217 12 12 0 0 0 0 0 0 0 + 0 0 0 0 0 0 10-'=-218 7 0 0 0 0 0 0 0 + 0 0 0 0 0 0 10-'=219 13 0 0 0 0 0 0 0 + 0 0 0 0 0 0 10-6=420 13 13 0 0 0 0 0 0 0 0 0 0 0 0 0000W-=421 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10-=422 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10-=223 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10'=424 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10-6-425 Control 9 + + + + + + 0 + + + + ++ 0 10-=326 A 8 + + + + + + 0 + + + + + + 0 lOz= 1027 6 + + + + + + 0 + + + + + + 0 107=328 13 + + + + + + 0 + + + + + + 0 I-'=329 1 + + + + + + 0 + + + + + + 0 10 =830 Control 2 + + + 0 0 0 0 + + + 0 0 0 0 10- = 231 B 12 + + 0 0 0 0 0 + + 0 0 0 0 0 10-=1032 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 10-=233 I 1 00 0 0 0 0 0 0 0 0 0 0 0 10-'=S

* + = growth.0 = no growth.

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GONOCOCCALINFECTION. I

TABLE II

Comparison of bactericidal power of whok defibrinated blood, unheated plasma, and heated plasma *

Observa- Patient Strain Whole blood Unheated plasma Heated plasma Num-tion num- numn -- - ---- ber of

10-1 10n- 10-5 10'-4 10- 10-| 16-7 10-110-D 10-8 10-' W-' 10-6 10-7 10-1 10-' 10-a 10-' 10-61 5J10-7 isms

1 5 + + 0 0 0 0 0 0 0 0 0 0 0 0 + + + + + ° ° 1°8-7

2 4 4 + + +OO O O+ + 0 )O 0+++++ ++ _1+ 0-79


body and complement in serum or plasma, thenheating either one to inactivate complement shouldresult in reducing the killing power. Table IIshows the results of heating plasma at 56° C. for30 minutes in a water bath and comparing itsbactericidal action with that of unheated plasmaand whole blood. Heated plasma had practicallyno bactericidal action on the gonococcus. Heatedplasma added to washed cells also resulted in thedeath of no organisms.

It was conceivable from these observations thatthe difference between the action of heated andunheated plasma was not due to the inactivationof complement but to the fact that unheatedplasma was a poor culture medium whereas heatedplasma was a good one. However, from the fol-lowing experiments, we demonstrated that un-heated plasma killed gonococci when antibodieswere present, and heated plasma failed to killthem owing to the inactivation of complement.

The first experiment showed that complementwas necessary for bactericidal action to take place.We obtained a polyvalent antigonococcal horseserum (Parke Davis) which agglutinated allstrains of gonococci used. This immune serumdid not contain complement. As a result, it isseen in Table III that the serum alone had no bac-tericidal power. However, when 0.1 cc. of fresh,undiluted human serum was added to 0.4 cc. ofthis immune serum, excellent bactericidal actionresulted. It is seen further that the same amountof fresh serum (0.1 cc.) diluted to 0.5 cc. did notkill the organisms. Only when the immune se-rum was activated by fresh serum containingcomplement did bactericidal action result.

To study further the bactericidal action in hu-man plasma, samples were obtained that possessedkilling power, and they were compared with those

TABLE IIIThe bactericidal action of immune serum with and without

the addition of fresh human serum (complement) *

Dilution of organism Num-ber of

10-1 10-' 10-5 10-4 10- 10" 10-1 Irs1. Immune rum 0.5 cc. 01c" + + + + + + + 10?

0.1 cc. 5

2. Freshhumanserum 0.lc. "C" + + + + + 0 0 10-I0.1 cc. 6

Normal salt solution 0.4

3. Immanesum0.4 co. IC" 0 0 0 0 0 0 0 10>0.1 cc. 6

Fresh hmansorum 0.1 ce.


showing no killing power. The results are re-corded in Table IV. Unheated plasma " W"

was capable of destroying the " C" strain ofgonococcus, whereas the heated serum had lostthis capacity. To test whether heating the serumdestroyed the antibody, immune serum was addedto the heated serum; no killing of the organismresulted. The heated serum was then reinacti-vated by the addition of fresh serum from a nor-mal individual, and the killing power was re-stored in part. From this experiment, it wouldappear that heating a plasma that was capable ofkilling gonococci inactivated complement and didnot destroy antibody. The addition of immuneserum to heated serum was ineffective in restor-ing bactericidal power, whereas the addition offresh serum (complement) restored it in part.

In the case of serum " K " which contained noantibodies against organisms " C " it was foundthat the addition of antibody (immune serum)caused killing when it was added to unheated se-rum, but no killing was observed when it wasadded to heated serum. The death of the or-ganisms was produced when immune serum was

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TABLE IV

The bactericidal action of heated and unheated human plasmawith and without antibodies *

Dilution of organismsObservations .o

10-1 10-2 10-3 10-4 10-6 10-6 107 10-7

Serum "W," unheated 0.5 + 0 0 0 0 0 0 5CC.

Organism " C" 0.1 cc.

Serum "W," heated 0.S cc. + + + + + + 0 5Organism " C" 0. 1 cc.

Serum "W," heated 0.4 cc. + + + + + + 0 5Organism "C" 0.1 cc. and

immune serum 0.1 cc.

Serum "W," heated 0.4 cc. + + + 0 0 0 0 5Organism "C" 0.1 cc. and

fresh serum "S" 0.1 cc.

Fresh serum "S" 0.5 cc. + + + + + + + 5and organism " C" 0.1 cc.

Immune serum 0.5 cc. and + + + + + + + 5organism " C" 0.1 cc.

Serum "D," unheated 0.5 0 0 0 0 0 0 0 2CC.

Organism "C" 0.1 cc.

Serum "D," heated 0.5 cc. + + + + + + + 2Organism " C" 0.1 cc.

Fresh serum "S" 0.5 cc. + + + + + + + 2and organism " C" 0.1 cc.

Serum "D," heated 0.5 cc. + 0 0 0 0 0 0 2Fresh serum "S" 0.5 cc.

and organism " C" 0.1 cc.

Serum "K," unheated 0.5 + + + + + + + 4CC.

Organism " C" 0.1 cc.

Serum "K," heated 0.5 cc. + + + + + + + 4Organism " C" 0.1 cc.

Serum "K," heated 0.5 cc. + + + + + + + 4Organism "C" 0.1 cc. and

immune serum 0.1 cc.

Serum " K," heated 0.25 cc. + + 0 0 0 0 0 4Immune serum 0.1 cc.,

organism "C" 0.1 cc. andfresh serum "S" 0.25 cc.

Serum " K. "unheated 0.5 cc. + 0 0 0 0 0 0 4Immune serum 0.1 cc.Organism "C" 0.1 cc.

* + = growth.O = no growth.

added to heated serum which had been reactivatedby fresh serum. The same results were obtainedwith another serum " D" which was obtainedfrom a patient with gonococcal arthritis.

It would appear to be established that the gono-coccus is killed in vitro by lysis. Both antibodyand complement are necessary for this action.

Bactericidal action of whole defibrinated bloodand washed cells suspended in saline

It has been recognized for many years that thegonococcus is actively phagocyted by polymorpho-

nuclear leukocytes. Whether or not this indicatesa method of destruction has remained in dispute.By a series of experiments we set out to deter-mine the relative importance of the cells in thedestruction of the gonococcus in vitro. In TableV, we present 5 observations comparing the bac-tericidal power of whole, defibrinated blood withthat of washed cells. The cells were washed 4times in sterile, physiological saline, as describedunder " Methods." It is plain from this experi-ment that the suspension of cells alone was notcapable of killing the gonococcus. When thecells were removed from the tubes, and smearswere made and stained, it was noted that activephagocytosis had taken place. There was, how-ever, no indication that bacterial growth had beensuppressed.

TABLE V

Comparison of bactericidal power of whole defibrinated bloodwith that of washed cells suspended in saline *

Obser- Whole defibrnated blood Washed cells in salinevation

nuimber10-11I" 10-' 10-410 10-5 10-7 10-' 10-10- 10-'10- ' 10-1

1 0 0 0 0 0 0 O + + + + + + 02 + 0 0 0 00 0 + + + + + + +3 0 0 0 0 00 0 + + + + + + 04t 0 0 0 0 0 0 0 + + + + 0 0 0

++++ 0 0 0 + + + + + + +


t No organisms were present in 6 and 7 dilutions.

A comparison of phagocytosis in specimens ofblood with and without bactericidal power

To supplement the above observations on phag-ocytosis and the r6le of the polymorphonuclearcells in killing the gonococcus, the degree of phag-ocytosis was studied in several cases. As hasbeen related in the bactericidal studies of thewhole blood, there was evidence that the gono-coccus was killed by lysis and not by intracellulardigestion. When the killing was complete, it wasnot possible to find any organisms within or out-side the cells. Inasmuch as it was not possible todemonstrate organisms when they were killed, thepossibility of intracellular digestion was not ab-solutely excluded. The experiment with cellsalone demonstrated that the organisms were en-gulfed by the leukocytes but growth was not sup-pressed, so that it seemed unlikely that the cellsactually killed them independently. It was ofinterest, however, to determine whether there was

172


TABLE VI

Comparison of bactericidal power and phagocytic activity of whole blood and washed cells from normal people andpatients with gonococcal arthritis *

po Phagocytosis whole Killing power Phagocytosis celswhole defibrinated blood defibrinated blood. ceUs washed in saline washed in saline.50 polys counted 50 polys counted

Observation

10-i 10'2 1013 10-4 10"' 10-6 10- WIt Wthu 10-i 10-2 1018 10-i 10-' 10'6 10' ih Wihuorganisms organisms organisms oraim

Patient I .... + + + 00 0 0 40 10 + + + + + + 0 49 1Control I .... 0 0 0 0 0 0 0 50 0 + + + + + + 0 45 5Patient 2 .... + + + + + + + 33 17Control 2 .... + + 0 0 0 0 0 30 20


any difference in the opsonic index of patientswith good bactericidal power and those with a

low titer.Table VI illustrates the killing power and the

phagocytic activity of the whole defibrinated bloodof patients with gonococcal infections as com-

pared with the results obtained with the blood ofnormal controls. It is to be noted that there isno correlation between the bactericidal power andphagocytic activity of the whole blood. The same

table also shows the absence of killing power ofcells washed in saline; a majority of the cells,however, engulfed organisms. Table VII givesa comparison of the killing power of whole de-fibrinated blood of normal controls with the de-gree of phagocytosis present.

It may be taken as established, then, that theopsonic index is no indication of the bactericidalpower of a patient's blood. Phagocytosis occurs

without killing, and the death of the bacteria canbe accomplished without cells.

The bactericidal effect of polyvalent antigonococ-cal horse serum when added to whole defibri-

nated blood of patients with gonococcalinfection

To determine whether the bacteriolytic power

of the whole blood could be enhanced by the addi-tion of immune serum, the following experimentwas done. A polyvalent antigonococcal horse se-

rum that agglutinated all strains of the gonococcus

that we studied was added to the whole defibri-nated blood of a patient with gonococcal arthritis.Table VIII illustrates the effect of adding 0.1 cc.

of a 1:10 and 1: 40 dilution of this serum to 0.5cc. of defibrinated blood of Patient C. The " C "

TABLE VII

Comparison of the killing power uith the phagocytic activityof whole defibrinated blood of normal controls *

Killing power whole blood polys countedObser-vationnum- With With-

10- 10" 10-1 10-' 10" 10" 107 organ- out

issisms1 + + + 0 0 0 0 40 102 ++ ++ + ++ 46 43 + + + + + + 0 37 134 + + + + + + 0 35 155 + + + + 0 0 0 44 66 + + + + 0 0 0 43 7


strain of gonococcus was obtained from this pa-tient, and it is seen that 0.5 cc. of his blood wascapable of killing only 7 organisms. Adding theabove dilutions of immune serum to his bloodgreatly increased the killing power. This proce-dure was repeated successfully several times using

TABLE VIII

Bactericidal power of whole defibrinated blood and that of thesame blood to which small amounts of gonococcal

immune serum had been added *

Whole Whole defibri- Whole defibri-Number of colonies defibrinated nated blood nated blood

"C " strain blood of +0.1 cc. +0.1cc.Patient "C" 1:10 immune 1:40 immune

serum serum

700,000 ......... + + +70,000 ......... + + 0

7,000 ......... + 0 0700 ......... + 0 0

70 ......... + 0 07......... 0 0 0


173


the blood of both normals and patients with gono-coccal infections. The antigonococcal horse se-rum did not contain complement and, therefore,had little or no killing power. Its bactericidal ac-tion depended upon the addition of fresh serumcontaining complement.

The bactericidal effect of whole defibrinated bloodof patients before and after the intravenous

administration of polyvalent antigono-coccal horse serum

As related above, it was found that when poly-valent, antigonococcal horse serum was added towhole defibrinated blood in vitro, the bactericidalpower of the blood was greatly increased. Wethen proceeded to administer the serum intra-

chart, were similar to those shown in the figure.When 0.1 cc. of a 1: 10 and 1:40 dilution of se-rum was added to the patient's blood in vitro, thekilling power was increased. By calculation itwas found that if 0.1 cc. of a 1:40 dilution ofimmune serum added to 0.5 cc. of his blood killedall the organisms except those in the first dilution,approximately 25 cc. of undiluted serum wouldhave to be administered intravenously to producethe same effect in vivo. Accordingly, 25 cc. ofpolyvalent, antigonococcal horse serum were ad-ministered intravenously without reaction. Fourhours later, a bactericidal test showed an increaseof the killing power of the patient's blood.Twenty-four hours later, the killing power hadreached the same level as that found when the

O IN VITRO0.1 CC.ANTI-G.C.

SERUM10 1:10 24 HRS.AFTER

1:40 SERUM

cc10 . 1:5-J -4 \

L&.3o + \10

zo0 -6P10

1025 CC. t t 4 HRS. AFTER

1 ANTI-G.C. SERUMSERUM

1 2 3 4 5 6 7 8 9 10DAYS

FIG. 1. THE BACTERIOLYTIC POWEROF THE WHOLEBLOOD FOLIMMUNESERUMin vitro AND in vivo.

venously to a patient for the purpose of determin-ing whether the same effect could be obtained invivo. The patient was recovering from an attackof acute gonococcal arthritis. The strain ofgonococcus was obtained from his prostatic dis-charge. In Figure 1, it is seen that the killingpower of the patient's blood was essentially thesame as that of a normal control. The results ofseveral other control tests, not included on the

15 20

LLOWING THE ADDITION OF

equivalent amount of serum was added in vitro.This maximum bactericidal action was of shortduration, however, and 5 days after receiving theserum, the killing power of his blood had returnedto the level observed before injection.

An opportunity arose for a practical demonstra-tion of the value of immune gonococcal serum ina patient with gonococcal septicemia. The focusof his infection was a chronic prostatitis; he did

174


not have an endocarditis. His illness was fur-ther complicated by cirrhosis of the liver withjaundice and ascites, and a hemorrhagic eruptionof the whole body due to a thrombocytopenia.Before the administration of immune serum,gonococci were cultured from his circulatingblood, and the killing power of his blood was lessthan that of the normal control. Immune serumwas then administered intravenously, and thefollowing day his blood was sterile, and there wasan increase in the titer of antibodies in the blood.On the basis of the preceding observation thatthis increase in bactericidal power resulting fromserum treatment may be of only short duration,daily injections of serum were administered.The patient's blood continued to show excellentkilling power. From these results, it seems ob-vious that the bactericidal power of the blood canbe enhanced in vivo and in vitro by the additionof antigonococcal immune serum. The thera-peutic significance of these observations will bediscussed in a subsequent paper.

DISCUSSION

From the evidence that we have presented,there can be little doubt that whole blood frommost patients with gonococcal infection is bacteri-cidal for the gonococcus. This property is afunction of the plasma or serum and it producesits action by means of lysis. Bacteriolysin andcomplement are necessary for this mechanism ofdestruction. There was no evidence that thepolymorphonuclear leukocytes were able to killgonococci in vitro. This was the case, in spite ofthe fact that active phagocytosis could be demon-strated. In this respect, it would appear that themechanism for the destruction of the gonococcusis similar to that of other gram-negative or-ganisms. Ward and Wright (2) in a similarstudy of the B. influenzae state, " Generally speak-ing, the gram-positive organisms are first sensi-

*tized by the antibody in the serum and then phag-ocyted by the leukocytes, no organisms being killedin the absence of the cells; but with the gram-negative organisms, the cells play a very minorrole, the bacteria being sensitized by the antibodyand then killed by the complement. Under cer-tain conditions the sensitized organisms are notonly killed, but undergo lysis. B. influenzae falls

into this category." Topley (3) is of the sameopinion in regard to such organisms as the choleravibrio, the typhoid bacillus, and "most gram-negative organisms." In an immunological studyof the meningococcus, which is closely related tothe gonococcus, Silverthorne and Fraser (4, 5)concluded that the plasma of human blood, andnot the cells, killed the meningococcus. The ear-lier work of Torrey (6) is of considerable signifi-cance in relation to immunological studies doneon guinea pigs inoculated with gonococci. Heconcluded that phagocytosis played little part inthe destruction of gonococci, since the degree ofphagocytosis was apparently the same whether theanimal died or recovered. Torrey believed thatthe rapid destruction of gonococci in the bloodwas due to specific bactericidal bodies. Furtherimportant observations were made by Martin (7)in studying the bactericidal action of an animal'sblood upon the gonococcus; he stated that comple-mented immune serum was most important in thedestruction of gonococci.

Although the circulating leukocytes, particu-larly the polymorphonuclear leukocytes, do notappear to play an important part in killing thegonococcus, it would be desirable to know moreconcerning the tissue cells and their action on thegonococcus. Torrey (6) studied the phagocyticactivity of cells in peritoneal exudates and of theomentum in guinea pigs injected intraperitoneallywith gonococci. He stated, that macrophages inthe peritoneal exudate ingested gonococci withmuch greater avidity than did the polymorphonu-clear leukocytes on the surface of the omentum.But the degree of phagocytosis was the samewhether the animal died or recovered.

Our information concerning the significance oftissue reactions in the prevention of spread ingonococcal infections is very meager. An earlyreaction to infections of the urethra is a profuseexudate consisting mostly of polymorphonuclearleukocytes. What action do these cells have onthe organism, especially those containing intra-cellular gonococci? If the organisms are notkilled when ingested by the leukocytes, it might bepostulated that this phagocytosis is an attempt tolocalize the gonococci and prevent their dissemi-nation throughout the tissues. Further immuno-logical studies are necessary before a proper eval-

175


uation can be made concerning the role of the tis-sue cells and those found in exudates in protectingthe body against widespread invasion by gono-cocci.

SUMMARY

1. A study of the bactericidal action of wholedefibrinated blood on several strains of gonococcijustifies the following conclusions.

a. Human defibrinated whole blood has theproperty of killing gonococci by means of lysis.

b. This bactericidal factor resides in serum orplasma, and is dependent upon the presence ofcomplement.

c. Heated serum or plasma, or immune serumwithout complement, have little or no bactericidalpower.

d. Freshly washed leukocytes suspended insaline have little or no killing action, though thepolymorphonuclear leukocytes actively phagocytethe organisms.

e. There does not appear to be any relationshipbetween the degree of phagocytosis and the bac-tericidal power of blood.

2. The addition of polyvalent, antigonococcalhorse serum to human blood greatly increases thekilling power.

3. The intravenous administration of immuneserum to patients greatly increases the killingpower of their blood.

We acknowledge our thanks to Miss Marjorie L.Jewell and Miss Eleanor M. Fleming for technical as-sistance.

BIBLIOGRAPHY

1. Spink, W. W., and Keefer, C. S., Studies of hemolyticstreptococcal infection. II. The serological reac-tions of the blood during erysipelas. J. Clin. In-vest., 1936, 15, 21.

2. Ward, H. K., and Wright, J., Studies on influenzalmeningitis. I. The problems of specific therapy.J. Exper. Med., 1932, 55, 223.

3. Topley, W. W. C., An Outline of Immunity. WilliamWood and Co., Baltimore, 1933, p. 73.

4. Silverthorne, N., and Fraser, D. T., The action of hu-man blood on the meningococcus. Brit. J. Exper.Path., 1934, 15, 362.

5. Silverthorne, N., and Fraser, D. T., Observations onthe action of human and animal blood on the me-ningococcus. J. Immunol., 1935, 29, 523.

6. Torrey, J. C., A study of natural and acquired immu-nity of guinea pigs to gonococcus. J. Med. Re-search, 1908, 18, 347.

7. Martin, W. B. M., The isolation of the gonococcus andits differentiation from allied organisms. J. Path.and Bact., 1910-11, 15, 76.

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