INFECTION AND IMMUNITY, JUlY 1970, p. 42-47 Vol. 2, No. ICopyright © 1970 American Society for Microbiology Printed in U.S.A.

Measurement of Candidacidal Activity of SpecificLeukocyte Types in Mixed Cell Populations

I. Normal, Myeloperoxidase-Deficient, and Chronic GranulomatousDisease NeutrophilsROBERT I. LEHRER

Cancer Research Institute and Department of Medicine, University of California School of Medicine,San Francisco, California 94122

Received for publication 17 March 1970

Candida albicans cells which survive ingestion and multiply within phagocytesdevelop characteristic filamentous pseudogerm tubes. Candida cells killed by phago-cytic leukocytes develop prominent alterations in Giemsa-staining characteristics;this reflects degradation of cyanophilic cytoplasmic components, probably ribo-nucleic acids. The numbers of these partially degraded organisms, termed "ghosts,"correlate closely with the percentage of Candida determined by an independentmethod to be nonviable. An assay, which makes use of these changes in morpho-logical and staining characteristics of ingested C. albicans, was developed to evaluatethe candidacidal activity of peripheral blood phagocytes. Neither myeloperoxidase-deficient neutrophils nor those from patients with chronic granulomatous diseasekilled C. albicans effectively, confirming observations made previously. Whereasmyeloperoxidase-deficient cells were able to retard the intracellular germination ofC. albicans, neutrophils from patients with chronic granulomatous disease lackedthis ability. The candidacidal activity of monocytes and eosinophils in small samplesof peripheral blood can also be measured by the new assay.

Leukocytes in the circulating blood constitutea major host defense mechanism against microbialinfection. The availability of methods to quanti-tate the bactericidal (3, 13) and candidacidal(10) activity of phagocytic cells has led to thedelineation of disorders, such as chronic granu-lomatous disease (16) and hereditary myeloperox-idase deficiency (11), wherein defective leukocytemicrobicidal function results in an increased sus-ceptibility to infectious disease.During investigations of the fate of Candida

albicans within leukocytes, the appearance of theingested fungi in fixed and stained slides wasnoted to correlate closely with their viability, asdetermined by differential staining (10) withaqueous methylene blue. The basis of the alteredstaining characteristics of nonviable organismswas investigated with purified enzyme prepara-tions, and a simple assay was constructed tomeasure the candidacidal activity of the varioustypes of phagocytic leukocytes in peripheralblood. In this report, the new assay is described,compared with previous methodology, and ap-plied to studies of normal and myeloperoxidase-deficient neutrophils and to neutrophils fromchildren with chronic granulomatous disease.

Other reports will describe its use in the charac-terization of the candidacidal activity of circu-lating human monocytes (9), and eosinophils.

MATERIALS AND METHODSC. albicans. All experiments utilized the UC 820

strain of C. albicans (10). Test organisms were grownin 50 ml of Sabouraud-2% dextrose broth (Difco) for72 to 120 hr at 33 C. The yeast-phase organisms werecounted in a hemocytometer, and their viability wasdetermined with methylene blue (10).

Leukocyte candidacidal assay. The "standard"leukocyte assay was performed as previously described(10). Briefly, 2.5 X 106 yeast-phase C. albicans andan equal number of neutrophils (added as mixedperipheral blood leukocytes) were incubated togetherat 37 C in a final volume of 1 ml of Hanks balancedsalt solution (HBSS) containing 25% normal groupAB serum. After 1 hr, the leukocytes were lysed byaddition of sodium deoxycholate, and the liberatedyeast cells were then exposed to a concentration ofmethylene blue (2 X 104 M) that stained only thenonviable yeast cells. This method will be referred toas the "dye-exclusion assay," and the use of methyleneblue to discriminate between nonviable and viableorganisms will be referred to as "dye exclusion."

Leukocytes and Candida for the "specific cell assay"were prepared as above. The final incubation mixture

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also contained 2.5 X 106 neutrophils and differedfrom that of the dye-exclusion assay onlv in that itcontained twice the number of C. albicans (5 X 106)in 1 ml. Control tubes contained Candida, serum, andHBSS. The tubes were incubated at 37 C in air withrotation (30 rev/min) for 2.5 hr. Duplicate slideswere then prepared from each tube by adding, with aPasteur pipette, one drop of the incubation mixtureand three drops of HBSS containing 15% fetal calfserum to the preparative chamber of a Cytocentrifuge(Shandon Scientific Co., London, England). Thechambers were rotated at 800 rev/min (about 50 X g)for 8 min, and the resulting slides were rapidly air-dried, fixed in absolute methanol for 1 min, andstained with Giemsa, which was prepared as a dilutionof 1 part of stock solution containing 1:1 glyceroland methanol (Uni-Tech, Sun Valley, Calif.) with 10parts of distilled water and used within 2 hr. Stainedslides were examined by light microscopy at 1,000-fold magnification, and 200 consecutive randomlyencountered Candida within phagocytic neutrophilswere classified as germinated yeasts, ungerminatedyeasts with blue cytoplasm, or "ghosts." "Ghosts"were ungerminated yeasts whose cytoplasm did notstain blue and which frequently had additionalmorphological abnormalities (see below).

Effect of enzymes on C. aibicans. Candida cells werewashed twice with distilled water, counted in a hemo-cytometer, and, where indicated, heat-killed in awater bath at either 70 or 100 C for 15 min. Ribo-nuclease-A (EC 2.7.7.16), five times crystallized andwith an activity of 90 Kunitz units/mg, and deoxy-ribonuclease-I (EC 3.1.4.5), once crystallized and withan activity of 2,250 Kunitz units/mg, both derivedfrom bovine pancreas, were purchased from SigmaChemical Co., St. Louis, Mo. Trypsin (EC 3.4.4.4),as a 2.5% solution, was purchased from GrandIsland Biological Co., Berkeley, Calif. Enzyme effectswere tested in 1-ml mixtures which contained 107heat-killed C. albicans, 20 Amoles of citrate-phosphatebuffer (pH 5 or 7), distilled water, and 0.2 mg ofribonuclease or deoxyribonuclease or 2.5 mg oftrypsin. After 2 and 4 hr, enzyme-treated Candidaand appropriate controls were examined for dyeexclusion and for Giemsa staining characteristics.

Differential heat inactivation of ribonuclease wasaccomplished, essentially as described by McDonald(14), by heating 2.0 mg of ribonuclease per ml indistilled water containing 0.02 M citrate-phosphatebuffer (pH 5) or 0.02 M phosphate buffer (pH 8) in awater bath at 100 C for 20 min. The heated enzymepreparations were rapidly cooled and then tested forribonuclease activity by the method of Kunitz (21)and for their effects on dye exclusion and Giemsastaining (see above).

Correlation between dye-exclusion and specific cellassays. Dye exclusion and Giemsa staining character-istics of ingested Candida were determined simul-taneously in 52 studies of neutrophils from normalsubjects, renal transplant recipients, and patients withvarious fungal and bacterial infections. Additionalstudies were performed on leukocytes from a 51-year-old man (C.J.B.) with hereditary myeloperoxidasedeficiency and two boys (R.T. and M.B.) with chronic

granulomatous disease. Case reports of these threepatients with hereditary leukocyte disorders haveappeared elsewhere (5, 11). At the time of study,C.J.B. was in good health and had not received ampho-tericin for more than 12 months. Although M.B. hadexperienced several episodes of relatively mild infec-tion during the preceding year, he was studied duringa disease-free interval that had lasted for severalmonths. Neutrophils of R.T. were tested duringhospitalization for a pyogenic liver abcess.

Standard statistical methods of comparison, in-cluding correlation and regression analysis, wereapplied to the data (18).

RESULTSIn methanol-fixed preparations of viable C.

albicans stained with Giemsa and examined bylight microscopy, the yeast-cell cytoplasm wascolored intensely blue, and the stain obscuredclear visualization of other intracellular struc-tures. If viable Candida cells that had germinatedin serum were similarly stained and examined,the cytoplasm within the spherical parent celland its slender germ tube appeared equally blue.When stained slides of leukocytes containingphagocytized C. albicans were prepared and theintracellular organisms examined, not only werethe blue-staining ungerminated and germinatingyeast cells recognized, but a class of organismswith a distinctive appearance was also ob-served. Still spherical in shape and lacking germtubes, these "ghosts" had a highly refractile cellwall as their single most prominent feature. Theircytoplasmic contents failed to take up the bluecolor and were, instead, either unstained or ofa rose-pink to pale-gray hue. A small, carmine-colored, eccentrically placed spherical structure,presumably the nucleus, was often discerned inthese "ghosts," particularly in slides prepared be-tween 1 and 2 hr after phagocytosis. In slides pre-pared 3 or 4 hr after phagocytosis, the cell wallshad lost much of their refractile quality, nuclearstructure was seldom as apparent, and the Can-dida remnants had sometimes swollen in size.

Effect of enzymes on staining of C. albicans. Inan earlier investigation, it was observed that, inunfixed preparations, heat-killed C. albicans wasstained by 2 X 10-4 M methylene blue, whereaslive organisms remained unstained (10). In thisinvestigation, we observed that after fixationboth heat-killed and live organisms were stainedintensely blue by Giemsa. Incubation of heat-killed C. albicans with trypsin or deoxyribonu-clease at pH 5 or 7 did not alter the respectiveaffinities of the organisms for methylene blue(unfixed preparations) or Giemsa (fixed slides).

In contrast, ribonuclease caused progressivechanges in the staining response of heat-killedorganisms to both Giemsa and methylene blue.

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FIG. 1. Staining of C. albicans by methylene blueand by Giemsa. At various times after incubation withmixed populations of peripheral blood leukocytes, thepercentage of C. albicans stained blue by 2 X 104 mmethylene blue (0) or appearing as "ghosts" inGiemsa-stained slides (0) was determined in deoxycho-late lysates. Each value is the mean from experimentson three normal subjects.

In the fixed, Giemsa-stained slides, many or-ganisms were uncolored or had cytoplasm whichvaried from pink to gray, closely resembling thealtered cytoplasmic staining in the phagocytized"ghost" cells. Although nuclear structure wasprominent in these cells, swelling or changes inappearance of the cell wall were not noted. Whenunfixed ribonuclease-treated cells were incubatedwith 2 x 10-4 M methylene blue, their cytoplasmstained less intensely than did that of the controlcells, and it sometimes displayed a greenish castinstead of the usual blue color.

Heating the ribonuclease preparation for 20min at pH 5 caused relatively little reduction inits ribonuclease activity and did not alter its ef-fects on the staining properties of heat-killedCandida. In contrast, ribonuclease heated for 20min at pH 8 lost virtually all ribonuclease activityand no longer caused nonviable Candida to betransformed into "ghosts."

Effect of time on staining. The candidacidal ac-tivity of mixed peripheral leukocytes was deter-mined in three normal subjects by the standarddye-exclusion assay. In addition, cytocentrifugedslides were made from a portion of the deoxy-cholate lysate prepared for viability counting inthe standard assay. After fixation and stainingwith Giemsa, the slides were examined and theCandida cels thereon were classified as germi-nated yeasts, ungerminated yeasts with blue cyto-plasm, or "ghosts" (ungerminated yeasts whosecytoplasm was not stained blue). Although many"ghosts" also displayed nuclear prominence orchanges in cell walls, the presence of such addi-tional features was not considered in making the

classification. Figure 1 compares the percentageof "ghosts" noted on Giemsa-stained slides withthe percentage of Candida considered nonviableby dye exclusion. It may be seen that the percent-age of nonviable cells as measured by dye exclu-sion rose rapidly, reached a plateau by 60 min,then declined gradually after 2 hr. In contrast,"ghosts" made their appearance more slowly.Their maximum rate of development occurredbetween 1 and 2 hr and thereafter rose very grad-ually. The percentage of Candida classified as"ghosts" between 2 and 3 hr after phagocytosisclosely approximated the percentage detected asnonviable by dye exclusion at 1 hr.

Staining of Candida within specific leukocytetypes. After various periods of incubation underthe conditions of the "specific cell assay," fixed,Giemsa-stained slides were prepared from mix-tures of normal leukocytes and C. albicans. Thepercentage of intracellular Candida that appearedas "ghosts" was determined separately for neu-trophils, monocytes, and, in some instances,eosinophils. In addition, deoxycholate lysates(see above) were prepared at each time period,and the percentage of "ghosts" therein was deter-mined on appropriately fixed and stained slides.In the experiment illustrated in Fig. 2, 98.5% ofthe added Candida cells were ingested by neutro-phils, 1.3% by monocytes, and 0.2% by eosino-phils. As might be expected from this distribution,the percentage of "ghosts" within neutrophils wasfound to be virtually identical to the percentageof "ghosts" in the deoxycholate lysate. However,the monocytes of this subject, although ingestingrelatively few of the added organisms, had anappreciably greater intrinsic ability to kill anddegrade ingested Candida than did his neutro-

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FIG. 2. Giemsa staining of Candida within specificleukocyte types. At various times after incubation withthe mixed peripheral blood leukocytes of a normalsubject, the percentage of Candida within monocytes(0) and neutrophils (A) that appeared as "ghosts"was determined. The open circles represent the per-centage of "ghosts" in lysates of the incubation mix-tures.

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FIG. 3. Comparison of dye-exclusion and specificneutrophil assays. The percentage of Candida stainedby 2 X 1J0-4 X methylene blue after I hr and thepercentage of Candida within neutrophils that were"ghosts" after 2.5 hr were determined in 52 experimentsotn normal subjects, renal transplant recipients, andpatients with various bacterial and fungal infections(Y = 0.93X + 1.96; r = 0.81).

phils. Eosinophils, although minimally phago-cytic, were intermediate between neutrophils andmonocytes in their ability to kill and degrade in-gested C. albicans (unpublished data).The pattern of more effective monocyte killing,

which emerged consistently in studies with nor-mal blood, appears to conflict with our earlierfinding that purified peripheral blood monocytes,studied by dye exclusion, exerted relatively inef-fective candidacidal activity (10). Additionalstudies (R. I. Lehrer, in preparation) revealedthat the prolonged and rigorous purificationprocedure employed in the earlier investigationscaused significant functional impairment of themonocytes, despite their continued viability andphagocytic activity.

Comparison of dye-exclusion and specific cellassays. In devising an assay based on the mor-phology of intracellular organisms, it soon be-came apparent that timing was a critical consid-eration. It was necessary to select an incubationperiod long enough for "ghost" formation toapproach maximal values in the phagocytic cells,yet short enough to ensure minimal disruption ofphagocytic cells by the rapidly enlarging pseudo-germ tubes of intracellular organisms that werenot killed. These considerations led to the adop-tion of a standard incubation time, 2.5 hr, thatmet both criteria. Neutrophils from 17 normalsubjects, examined after the 2.5-hr incubationperiod, were found to contain 28.1 ±i 7.6%(mean ±fi 1 standard deviation) of ingested Can-dia as "ghosts," 56.8 i 7.2% as ungerminated

yeasts with blue cytoplasm, and 15.1 ± 6.8%o asgerminated yeasts.The candidacidal activity of mixed peripheral

blood leukocytes as determined by the dye-exclu-sion assay reflects, almost exclusively, neutrophilfunction (10). The percentage of Candida consid-ered nonviable by dye exclusion 1 hr after inges-tion by peripheral blood leukocytes was thereforecompared with the percentage of Candida withinneutrophils that were "ghosts" after 2.5 hr(Fig. 3). There was a high degree of correlation(r = 0.81) between the two measurements ofcandidacidal activity, and the slope of the line ofregression was not significantly different fromunity (0.4 > P > 0.2).

Hereditary disorders of leukocyte function.Studies with the standard dye-exclusion assay in-dicated that neutrophils from a patient with he-reditary myeloperoxidase deficiency and fromchildren with chronic granulomatous disease weredefective in their ability to kill ingested C. albicans(10, 11). These findings were confirmed by use ofthe specific cell assay in the present studies(Fig. 4). However, a noteworthy difference in thebehavior of C. albicans within neutrophils frompatients with these two hereditary disorders wasobserved. Both normal and myeloperoxidase-de-ficient cells retarded the germination of a signifi-cant fraction of the ingested organisms that werenot "ghosts." Thus, after 1 hr of incubation, only0.8 ±i 1.2% of yeasts in normal neutrophils(six studies) and 4.5 + 4.8% of yeasts in myelo-peroxidase-deficient neutrophils (C.J.B., fourstudies) had formed pseudogerm tubes. In neutro-

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by human neutrophils. After incubation for the indicatedtimes, morphology of intracellular yeasts was deter-mined in fixed, Giemsa-stained slides. Symbols: 0,neutrophils from seven normal subjects; 0, repeatedstudies of myeloperoxidase-deficient neutrophils fromC.J.B.; A, neutrophils from lwo children with chronicgranulomatous disease (R.T. andM.B.);A A, the almostcomplete disruption of the chronic granulomatous dis-ease neutrophils by the germinated Candida.

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phils from R.T., a patient with chronic granulo-matous disease, 32% of the phagocytized organ-isms had germinated within 1 hr; this was insignificant contrast to the behavior of Candidawithin either normal (P < 0.001) oI myeloper-oxidase-deficient (P < 0.025) neutrophils. Neu-trophils from M.B., the other patient withchronic granulomatous disease, were even lesseffective than normal (P < 0.001) or myeloperox-idase-deficient (P < 0.005) neutrophils in inhibit-ing germination. As an additional check duringthese observations, the neutrophils of M.B. werestudied simultaneously with normal and myelo-peroxidase-deficient cells, confirming that thelarge differences in the percentage of germinatedCandida observed 1 hr after phagocytosis (M.B.,63%; C.J.B., 9.5%; normal, 3%) reflected thedeficient candidastatic ability of his neutrophilsrather than the effect of variations in experimen-tal conditions. In fact, the Candida phagocytizedby neutrophils from the patients with chronicgranulomatous disease germinated about asreadily as did Candida incubated with serumalone.

DISCUSSIONThe observation of Metschnikoff that phago-

cytized microorganisms developed alterations oftheir morphological and staining properties andhis inference that this reflected microbial deathand degradation within the phagocyte (15) werethe basis of the work by Bordet, who reported in1895 that intracellular bacteria stained with mix-tures of eosin and methylene blue initially ap-peared blue but gradually assumed a pink coloras a consequence of events within the phagocyte(1). It is noteworthy that Giemsa stain, a mixtureof eosin, methylene blue, and polychromed meth-ylene blue (4), was employed in the present study.Giemsa-staining characteristics, analogous tothose described here for C. albicans, have previ-ously been reported to be a valuable means ofdistinguishing live from killed Histoplasma cap-sulatum (7) and Dermatophilus congolensis (17)within phagocytic leukocytes.

Unlike our earlier methylene-blue exclusionmethod, we found that Gie,msa-staining charac-teristics did not correlate with the viability ofC. albicans per se. However, by detecting organ-isms whose cytoplasm had been depleted ofcyanophilic constituents, presumably ribonucleicacid (RNA), after their death within phagocytes,Giemsa stains provided an indirect marker ofcellular candidacidal activity. In radioisotopicstudies of the kinetics of degradation of the mac-romolecular constituents of phagocytized bac-teria, Cohn observed bacterial RNA to be morereadily degraded than deoxyribonucleic acid (2).

Our observation that alterations in cytoplasmicstaining compatible with depletion of RNA reg-ularly preceded the disappearance of the pre-sumed nuclear structure in killed C. albicanssuggests that fungi may be similarly affected byphagocytes.The rapid assumption of a filamentous growth

pattern by live yeast-phase C. albicans in serum(20) or cells facilitates a morphological approachto the analysis of their survival within phago-cytes. Stanley and Hurley used this criterion toestablish that peritoneal macrophages were in-capable of killing ingested C. albicans (19).Louria and Brayton made similar measurementsin a study of the survival of C. albicans withinneutrophils. They made the interesting observa-tion that the pathogenicity for mice of C. albicansstrains broadly paralleled their ability to surviveand germinate within neutrophils (12).

In the present study, the rate at which filamen-tation occurred disclosed a functional differencebetween myeloperoxidase-deficient human neu-trophils and those from two patients with chronicgranulomatous disease. In the former condition,neutrophils lack the lysosomal enzyme myelo-peroxidase but develop a normal postphagocytic"burst" of oxygen consumption (11). In the lattercondition, neutrophils possess myeloperoxidasebut are deficient in their oxidative response tophagocytosis and consequently fail to generateintracellular hydrogen peroxide (6). That neithertype of neutrophil was effective in killing C. albi-cans supports the hypothesis that both myelo-peroxidase and hydrogen peroxide are requiredfor candidacidal activity (8). However, in contrastto chronic granulomatous disease neutrophils,myeloperoxidase-deficient cells exerted a demon-strable candidastatic effect, manifested by thedelayed filamentation of the ingested organisms.This suggests that the oxidative, postphagocyticmetabolic response of neutrophils can, in theabsence of myeloperoxidase, exert an inhibitoryeffect on ingested fungi. Although this inhibitionhad but a limited effectiveness against C. albicans,other fungi could conceivably be more susceptibleto its action.The assay described in this report affords a

simple way to measure the candidacidal activityof the phagocytes in peripheral blood. When ap-plied to neutrophils, it provides essentially thesame data that can be obtained from the previ-ously described dye-exclusion method (10). Itsspecial utility arises from its smaller blood re-quirement (less than 5 ml) and from its uniqueability to allow study of the microbicidal activityof monocytes and eosinophils at the same timethat neutrophil function is evaluated without re-quiring additional purification procedures. It may

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therefore contribute to the elucidation of theroles played by various phagocytic cell types inresistance to Candida infection.

ACKNOWLEDGMENTS

I thank Gisela Schanklies for her skillful technical assistance.This work was supported by Public Health Service research

grant CA-1 1067 from the National Cancer Institute and by CancerResearch funds from the University of California.

LITERATURE CITED

1. Bordet, J. 1895. Contribution a l'etude du serum chez lesanimaux vaccines. Ann. Soc. Roy. Sci. Med. Nat. Bruxelles4:455-530.

2. Cohn, Z. A. 1963. The fate of bacteria within phagocytic cells.I. The degradation of isotopically labelled bacteria by poly-morphonuclear leucocytes and macrophages. J. Exp. Med.117:27-42.

3. Cohn, Z. A., and S. I. Morse. 1959. Interactions between rab-bit polymorphonuclear leucocytes and staphylococci. J.Exp. Med. 110:419-443.

4. Conn, H. J. 1953. Biological stains. A handbook on the na-

ture and uses of the dyes employed in the biological labora-tory, 6th ed. rev., p. 244. Biotech Publications, Geneva,N.Y.

5. Douglas, S. D., W. C. Davis, and H. H. Fudenberg. 1969.Granulocytopathies: pleomorphism of neutrophil dysfunc-tion. Amer. J. Med. 46:901-909.

6. Holmes, B., A. R. Page, and R. A. Good. 1967. Studies of themetabolic activity of leukocytes from patients with a geneticabnormality of phagocytic function. J. Clin. Invest. 46:1422-1432.

7. Howard, D. H. 1964. Intracellular behavior of Histoplasmacapsulatum. J. Bacteriol. 87:33-38.

8. Lehrer, R. I. 1969. Antifungal effects of peroxidase systems. J.Bacteriol. 99:361-365.

9. Lehrer, R. I. 1970. The fungicidal activity of human mono-

cytes: a myeloperoxidase-linked mechanism. Clin. Res. 18:408.

10. Lehrer, R. I., and M. J. Cline. 1969. Interaction of Candidaalbicans with human leukocytes and serum. J. Bacteriol.98:996-1004.

11. Lehrer, R. 1., and M. J. Cline. 1969. Leukocyte myeloperoxi-dase deficiency and disseminated candidiasis: the role ofmyeloperoxidase in resistance to Candida infection. J. Clin.Invest. 48:1478-1488.

12. Louria, D. B., and R. G. Brayton. 1964. Behavior of Candidacells within leukocytes. Proc. Soc. Exp. Biol. Med. 115:93-98.

13. Maaloe, 0. 1946. On the relation between alexin and opsonin.Einar Munksgaard, Copenhagen, Denmark.

14. McDonald, M. R. 1948. Proteolytic contaminants of crystal-line ribonuclease. J. Gen. Physiol. 32:33-37.

15. Metschnikoff, E. 1887. Sur la lutte des cellules de l'organismecontre l'invasion des microbes. Ann. Inst. Pasteur (Paris)1:321-326.

16. Quie, P. G., J. G. White, B. Holmes, and R. A. Good. 1967.In vitro bactericidal capacity of human polymorphonuclearleukocytes: diminished activity in cbronic granulomatousdisease of childhood. J. Clin. Invest. 46:668-679.

17. Roberts, D. S. 1966. The phagocytic basis of acquired re-

sistance to infection with Dermatophilus congolensis. Brit. J.Exp. Pathol. 47:372-382.

18. Snedecor, G. W., and W. G. Cochran. 1967. Statistical meth-ods, 6th ed. Iowa State University Press, Ames, Iowa.

19. Stanley. V. C., and R. Hurley. 1969. The growth of Candidaspecies in cultures of mouse peritoneal macrophages. J.Pathol. 97:357-366.

20. Taschdjian, C. L., J. J. Burchall, and P. J. Kozinn. 1960.Rapid identification ofCandida albicans by filamentation on

serum and serum substitutes. Amer. J. Dis. Child. 99: 212-215.

21. Z6l1ner, N., and G. Hobom. 1965. Ribonuclease, p. 793-799.In H.-U. Bergmeyer (ed.), Methods of enzymatic analysis.Academic Press Inc., New York.

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