Leukocyte Degranulation and Vacuole

Formation in Patients with Chronic

Granulomatous Disease of Childhood

EMANUELKAUDER, Louis L. KAHLE, HERNANMORENO,andJOHNC. PARTIN

From the Department of Pediatrics, University of Cincinnati, The Children'sHospital, and the Children's Hospital Research Foundation,Cincinnati, Ohio 45229

A B S T R A C T A cellular defect associated withdecreased bactericidal activity of the polymorpho-nuclear leukocyte has been found in a 2% yr oldNegro boy with the typical clinical and pathologi-cal findings of chronic granulomatous disease.Unlike previously described patients his poly-morphonuclear leukocytes were shown to undergoapparently normal degranulation and vacuoleformation after phagocytosis. Metabolic studies ofthe leukocytes indicated a failure to increase oxy-gen consumption with phagocytosis or to reduceNitroblue tetrazolium dye. These metabolic ab-normalities are identical with those previously re-ported in patients with chronic granulomatousdisease. Two additional patients with chronicgranulomatous disease have also been found tohave apparently adequate degranulation of poly-morphonuclear leukocytes after phagocytosis.Our studies suggest that failure of degranulationmay not be a necessary part of this functional leu-kocyte abnormality.

INTRODUCTION

Among the chronic granulomatous diseases ofchildren is a group of strikingly similar patients

This work was presented in part at the 37th meetingof the Society for Pediatric Research, Atlantic City, 29April 1967.

Address requests for reprints to Dr. E. Kauder, TheChildren's Hospital, Cincinnati, Ohio 45229.

Received for publication 7 July 1967 and in revisedform 8 February 1968.

that were first brought together as a clinical en-tity under the name of "a fatal granulomatousdisease of childhood" (1). The polymorphonuclearleukocyte (PMN) of these patients has beenshown to have decreased bactericidal activity by invitro measurement (2). Recent reports have fo-cused attention on morphologic and metabolic ab-normalities in the functionally abnormal PMN.In the normal PMNdegranulation and vacuoleformation occur after phagocytosis (3). Quie,White, Holmes, and Good (4) have reported afailure of these events to occur in the PMNof pa-tients with chronic granulomatous disease (CGD).Holmes, Page, and Good have reported that anacid extract of the PMNhad normal bactericidalactivity in vitro (5). Baehner and Nathan (6)reported a failure to reduce Nitroblue tetrazolium(NBT) of leukocytes from two patients withCGD. The hexose monophosphate shunt (HMS)also failed to be normally stimulated during phago-cytosis. In one patient they measured oxygenconsumption of the leukocytes during phagocyto-sis and found that it was not increased. They at-tributed these metabolic abnormalities to the lackof a cyanide-insensitive nicotinamide adenine di-nucleotide (NADH) oxidase.

Holmes-et al. (5) have recently reported on ex-tensive metabolic studies in leukocytes of patientswith CGD. They concluded that glucose utilization,lactate production, Kreb's cycle activity, and lipidturnover all were stimulated normally duringphagocytosis. However, respiration, hydrogen

The Journal of Clinical Investigation Volume 47 1968 1753

peroxide production, and HMSactivity were notnormally increased with phagocytosis. They pos-tulated that these impaired metabolic events arerelated to a failure of degranulation.

However, Zatti, Rossi, and Meneghelli (7) havestudied the time of stimulation of the HMSrela-tive to the time of degranulation in phagocytosingleukocytes and reported that increased HMSac-tivity occurred before the act of degranulation,suggesting to these authors that the stimulation tooxidative glycolysis was not due to degranulationbut to the act of particle ingestion by the cell.

Holmes and coworkers (5) have also challengedthe concept, put forth by Baehner and Nathan, of alack of NADHoxidase in these abnormal leuko-cytes. Obviously this degree of controversy indi-cates that more needs to be learned about the meta-bolic abnormality in the leukocyte of the patientwith CGD.

The purpose of this report is to present evidencethat degranulation and vacuole formation mayoccur normally in a patient who fulfills all thecriteria for CGD.

METHODSCase Report. The patient was a 2A yr old Negro boy

who had his first onset of cervical adenitis at 3A monthsof age. Recurrent adenitis required incision and drainageon five subsequent occasions. Seborrheic eczema of thescalp and neck with secondary pyoderma began at 12months of age. Pulmonary disease with recurrent pneu-monia and' persistent pulmonary infiltrates has beenpresent since 15 months of age. Other bacterial infectionshave included an hepatic and abdominal wall abscess andan iliopsoas abscess. Moderate hepatosplenomegaly hasbeen present intermittently at times of infection. Thereare seven older siblings, three of whom are male. Noneof these family members had a significant history ofrepeated infections.

Laboratory findings. Appropriate leukocytosis occurredat the time of bacterial infections with absolute neutrophilcounts ranging from 4000 to 25,000/mm3. Hemoglobinconcentration was 9 g/100 ml with hypochromia andmicrocytosis. Hyperglobulinemia with elevations of IgA,IgM, and IgG was first noted at 15 months of age andhas been persistent since. Leukocytes entered into areasof inflammation normally as demonstrated by a normalresponse to "Rebuck skin window" (8) preparations andthe presence of abundant leukocytes in purulent materialfrom abscesses. Normal delayed hypersensitivity wasdemonstrated by a positive mumps skin test. An excisionalbiopsy of an inguinal node was done and a caseatedgranuloma found. Skin tests for tuberculosis, histoplas-mosis, and atypical mycobacteria were repeatedly negativeas were cultures for these organisms from biopsy material

and abscesses. Cultures f rom abscess material yieldedStaphylococcus aureus and Aerobacter aerogenes.

Phagocytosis studies. Phagocytosis and bactericidalproperties of intact leukocytes were determined by amodification of the method described by Maal0e (9) withfurther modifications as described by Cohn and Morse(10). Leukocyte suspensions were prepared by dextransedimentation of heparinized venous blood. 20 ml offreshly drawn blood was mixed with 40 ml of 3% steriledextran (mol wt 228,000) containing 100 U of sterileheparin, and allowed to sediment at 37°C for 20-30 min.The supernatant plasma layer containing the leukocyteswas harvested and spun in sterile siliconized graduatedcentrifuge tubes for 10 min at 500 g (1500 rpm, PR-2International centrifuge, head No. 269). The cell buttonswere washed two times with warm Hank's solution. Thewhite cell concentration was determined by hemocytometercounting, smears were stained with Wright's stain, anddifferential counts were performed. Cell suspensions werethen diluted to give a PMNconcentration of -1-3 X 107/ml.Appropriate dilutions were calculated to give approxi-mately the same number of neutrophils per milliliter forpatient and control samples.

The bacterial species used in these studies were Staph-ylococcus aureus strain 502A and Aerobacter aerogenes,The Staphylococcal strain has previously been describedand its biological characteristics defined (11). The A.aerogenes species was isolated from drainage from anileopsoas abscess from the patient. The organism to beused in each study was cultured overnight in trypticaseSoy Broth at 37°C for 18 hr. The bacterial suspensionwas then centrifuged and washed two times with Hank'ssolution in a manner similar to the cell suspensions. Afterthe second wash, the bacterial button was resuspendedin an equal volume of Hank's solution and the concentra-tion of organisms determined using a Petroff-Hausserbacterial counter. The bacterial concentration was thenadjusted with Hank's solution to give approximately1 X 108 bacteria per ml.

As a source of serum opsonins for the phagocytosismixture fresh blood was obtained at the same bloodletting utilized for obtaining the blood for leukocyteisolation and allowed to clot at room temperature. Indifferent experiments, the serum obtained from the clottedblood was collected from either the patient or thecontrol donor.

Phagocytosis studies were done in sterile siliconized30-ml French bottles. Each bottle contained 1.6 ml ofthe cell suspension, 0.2 ml of serum, and 0.2 ml of thebacterial suspension. This resulted in an approximatelyequal ratio of bacteria to leukocytes and a final concen-tration of 10% serum.

Immediately after the suspensions were made theywere thoroughly mixed using a vortex mixer and a0.1 ml aliquot was obtained from each bottle, mixed with0.9 ml of saline, and each sample was homogenized for34 min using a teflon homogenizer driven by a highspeed motor. Serial dilutions with normal saline weremade and 0.1 ml of a 1/10,000 dilution was mixed withmelted Trypticase Soy Agar and pour plates made to de-

1754 E. Kauder, L. L. Kahle, H. Moreno, and J. C. Partin

termine the total number of viable organisms. The leuko-cyte-bacterial mixtures were then incubated at 370C ina temperature-controlled water bath shaker, set at 30agitations/min. At 1 and 2-hr intervals another 0.1 mlaliquot was obtained and processed in the same mannerfor determining total viable organisms. At the same time,a 0.5 ml aliquot was obtained from each bottle anddiluted with 4.5 ml of normal saline. This was centrifugedat 600 rpm for 4 min. The supernatant containing theextracellular bacteria was removed and a pour platemade from a 0.1 ml aliquot of a 1/10,000 dilution. Thecell button containing the intracellular bacteria was re-constituted to 0.5 ml in normal saline. A 1/10 dilutionwas homogenized to release intracellular bacteria and a1/10,000 dilution was prepared from a 0.1 ml aliquot ofthe homogenate. Pour plates were prepared as above.The colonies were counted on the following day afterovernight incubation at 370C and total, extracellular, andcell-associated viable bacteria determined for each incu-bation period.

Wright's-stained cover slip smears were also obtainedbefore incubation and at each incubation period fordetermination of percentage of PMN participating inphagocytosis, the average number of bacteria per phago-cytosing PMN, and the degree of degranulation andvacuole formation. Cover slip smears were also preparedand stained for alkaline phosphatase and peroxidase. Wetpreparations of leukocyte-bacterial mixtures were exam-ined under phase microscopy and leukocyte motility andphagocytosis observed.

Preparation for electron microscopy. Leukocyte-bac-terial suspensions were fixed in 3% glutaraldehyde inMillonig's phosphate buffer at pH 7.35. They were post-fixed in 1% osmium tetroxide in the same buffer andthen embedded in araldite. The tissue was sectioned withglass knives and examined using Zeiss EM 9 and Philips300 electron microscopes. Sections were stained with leadand uranyl acetate. Some were lightly shadowed withcarbon.

Procedure for determining oxygen consumption. Leu-kocytes were separated by dextran sedimentation as de-scribed above. No attempt was made to obtain red bloodcell-free suspensions. Cells were washed in Krebs-Ringerphosphate buffer and resuspended in 1 ml of this buffer.Equal numbers of neutrophils for. the patient and controlstudies were utilized for each experiment and suspensionswere adjusted to contain between 2 and 4 X 107 cells/ml.A glucose solution was prepared in Krebs-Ringer phos-phate buffer containing 1 mmole of glucose per ml. Justbefore the start of each experiment, 1 ml of glucose solu-tion was added to 1 ml of cell suspension. The sample tobe tested was placed in an ultramicrosample system(model No. 113-Si Instrumentation Laboratory, Inc.,Boston, Mass.).

We allowed 5 min for equilibration and then tookreadings every 5 min for 20 min, mixing the sample afterevery reading. This constituted the resting state. 2/10 mlof polystyrene latex particles (Difco, 0.81 u) in suspen-sion was then added through a three-way stopcock andafter thorough mixing, readings were taken every 3 min

for 20 min. The hemoglobin in each sample was thendetermined and glass cover slip smears obtained andstained with Wright's stain to ascertain phagocytosis.The partial tension of oxygen was measured polarographi-cally. Since all samples were maintained at 370C notemperature correction factor was necessary. Serial sam-ples were withdrawn for determination of pH.

The pH of the cell suspension decreased during theperiod of sampling. The measured oxygen tension wascorrected for pH change by correction factors obtainedfrom Severinghaus (12) and converted to saturation inper cent by a nomogram (12). The oxygen carryingcapacity of the hemoglobin in these samples was calcu-lated from hemoglobin content measured by the cyan-metheglobin method (13) multiplied by the factor. 1.34(14). The oxygen carrying capacity was multiplied bythe saturation to obtain the oxygen content in volumesper cent for each sample. The difference in oxygen con-tent from the time of initial measurement to that of thelast sample was taken as a measure of oxygen consump-tion by the leukocytes in the sample.

Cellular Uptake of p-Nitroblue tetrazoliuint chloride(NBT). NBT uptake and reduction intracellularly inleukocytes participating in phagocytosis was studied usingthe method of Windhorst, Holmes, and Good (15).Leukocyte-rich plasma was obtained from 10 ml ofheparinized venous blood by sedimentation with clinicaldextran. Leukocyte counts were done on the supernatantplasma and an aliquot calculated to contain 7 X 106 cellswas transferred to a siliconized test tube and centrifugedat 500 rpm. The cells were resuspended in 0.35 ml of nor-mal human serum to which 0.16 mg of glucose in 0.05 mlof water was added to ensure adequate oxidative sub-strate. 3 mg of nicotinamide adenine dinucleotide phos-phate (NADPH) and 0.13 mg of NBT in 0.1 mg ofphosphate buffer at pH 7.0 were added to the cell sus-pension which was mixed and allowed to stand at 37°Cfor several minutes. Latex particles (Difco, 0.81 ) werethen added and the tubes were gently mixed on a tilttable at 37°C for 45 min. The cells were then centrifugedat 500 rpm. Smears were prepared from the cell buttonand stained with neutral red. Only cells containing 10 ormore latex particles were counted. These were counted aspositive if a blue precipitate could be seen in the cyto-plasm. This method was modified from that of Wind-horst and coworkers, in that the cells were incubated withNBT for 45 min and all reagents were kept at 37°C.These modifications produced more consistent results withnormal cells which labeled to 70%o or more.

Morphology and cytochemistry of leukocytes. Bloodsmears and smears of leukocyte-bacteria mixtures weremade on glass slides and stained with Wright's stain instandard fashion. Slides were prepared for histochemicaldetermination of peroxidase by the method of Graham(16) and for alkaline phosphatase by the method ofAckerman (17).

RESULTS

Phagocytosis. The results of the study to mea-sure intracellular killing of ingested bacteria are

Chronic Granulomatous Disease of Childhood 1755

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Time of incubationFIGURE 1 Location and number of viable bacteria after1 and 2 hr incubation of leukocyte-bacteria mixtures.Note that most of the viable bacteria were cell associatedwith many more viable organisms in the patient's leuko-cytes than in the control cells. Organism, Staphylococcusaureus 502A; serum, control; black bars, extracellularbacteria; striated bars, cell associated bacteria.

shown in Fig. 1. There was a striking persistenceof viable cell-associated organisms in the patient'sstudy as compared with the control. The controlcells performed normally in other similar experi-ments when the patient's serum was used as asource of opsonins. The patient's cells were de-fective against both Staphlvlococcits alfreats andAcrobacter aerogencs.

Light microscopy. In Wright's-stained bloodsmears examined at several intervals after incuba-tion of leukocyte-bacterial suspensions there wasan equal degree of degranulation and vacuole for-mation for the patient's and for control leukocytes.There was no significant difference in the per-centage of PMNcontaining ingested bacteria, the

average number of bacteria per PMN, or the per-centage of PMNin which bacteria were containedwithin vacuoles (Table I). However, in the con-trol cells a larger percentage of bacteria appearedto be undergoing fragmentation and digestion ascompared with a higher percentage of intact bac-teria in the cells of the patient. To assess the de-gree of degranulation in these leukocyte-bacterialsuspensions peroxidase and alkaline phosphatasestains were used to highlight the granular miorphol-ogy. At 5, 30, and 60-min incubation times, slideswere prepared with peroxidase and alkaline phos-phatase stain, coded, and read by a single observer.200 leukocytes containing one or more bacteriawere counted per slide and the degree of degranu-lation recorded as minimal, moderate, or marked.No difference in degranulation could be detectedbetween normal cells and those of the patient.

These studies were performed in two additionalpatients with chronic granulonmutotus disease.Their diagnoses were based upon the typical clini-cal features of the syndrome plus the characteristicfunctional and metabolic abnormalities of the poly-morphonuclear leukocytes as described for the firstpatient. In one of these children the typical granu-lomatous lesion was demonstrated in a biopsy ofan area of chronic suppurative lymphadenitis. Asseen in Fig. 2 a and b the patients' PMNcontainedvacuoles and a decreased number of cytoplasmicgranules after phagocytosis.

Active motility directed towards phagocytosisand followed bv (legranulation and vacuole forma-tion was seen wxith phase microscopy.

Electron ni icroscopy. Electron photomicro-graphs were prepared from suspensions of the

TABLE I

Phagocytosis and Vacuole Formation

Stapyhlococcus aureus A. Aerogenes

Organism, incubation period Patient Control Patient Control Patient Control Patient Control

I hr 2 hi I hi 2 hr

'/c PMNwith bacteria 43 37 54 45 60 32 87 63

Average No. of bacteria 4 4 4 4 5 3 8 4per PMN

%PMNwith bacteria 91 92 94 95 94 98 94 92having vacuoles

PMN, polymorphonuclear leukocyte.

1756 E. Kauder, L. L. Kahle, H. Moreno, and J. C. Partin

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FIGURE 2 a and b Light microscopy photographs of peroxidase stained 1 hr leukocyte-bacteria mixtures. Thesephotomicrographs from studies of two additional patients with chronic granulomatous disease include cells whichhave not ingested bacteria (Staphylococcus aureus 502A) in which granules are intact and no vacuoles are seen. Incontrast most cells have undergone marked degranulation and vacuole formation. X 2000.

patient's leukocytes after 15 and 60-min incuba-tion with Aerobacter aerogenes. Approximately500 leukocytes containing bacteria were examinedfrom each of these time periods. At the 15 min in-cubation time bacteria could be seen within thecells but cytoplasmic granules were numerous anddigestive vacuoles were small (Fig. 3). At the 1hr incubation time digestive vacuoles were wellformed and the number of cytoplasmic granuleshad decreased (Fig. 4). A higher magnification ofthe digestive vacuole is shown in Fig. 5 to demon-strate the clear evidence of granular fragmentsand whole granules emptying into a vacuole fromthe surrounding cytoplasm. We could detect nodifference in the time sequence or the degree towhich these events occurred in normal leukocytesstudied under the same conditions. A consistentdifference between the control and the patient

studies was the presence of a higher percentageof intact appearing bacteria in the latter.

Nitroblue tetrazolium study. The number ofphagocytic leukocytes containing NBT dye variedfrom 0-15% in the patient's cells. The patient'smother and two female siblings had values rangingfrom 26-66%. The range of normal in our labora-tory is 70-98% (mean 85%). Three male siblingsand two other female siblings had normal NBTstudies.

Oxygen consumption study. As shown in Fig.6, the patient's leukocytes failed to show an in-crease in oxygen consumption during phagocytosisas compared with normal cells. This demonstrationof failure to stimulate respiration with phagocyto-sis is presented as additional evidence that our pa-tient is similar to those described by Holmes andcoworkers (5) and by Baehner and Nathan (6).

Chronic Granulomatous Disease of Childhood

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FIGURE 3 Patient's polymorphonuclear leukocyte incubated 15 min with bacteria. Two bacteria (b) are withinphagocytic vacuoles. Numerous granules (arrows) are present. N, nucleus; G, golgi zone. X 24,000.

1758 E. Kauder, L. L. Kahle, H. Moreno, and J. C. Partin

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FIGURE 4 Patient's polymorphonuclear leukocyte incubated 1 hr with bacteria. The degree of degranulation issimilar to that seen in the normal control leukocytes. Cytoplasmic granules are decreased in number as com-pared with the 15 min incubation time. The bacterial bodies (b) show less evidence of disruption than was seenin controls. Cytoplasmic granules are adjacent to and within the digestive vacuole (arrows). N, nuclear lobes; G,golgi zone. X 24,700.

Chronic Granulomatous Disease of Childhood 1759

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A:~~~~~~~~~~FIGURE 5 Higher magnification (ca. 0000) of digestive vacuole in patient's polymorphonuclear leukocyte after 1hr incubation with bacteria. This typical digestive vacuole contains five undigested bacterial bodies. Lysomal gran-ules (arrows) are adjacent to, entering, and within the vacuole. N, nucleus; M, Mitochondria. X 37,000.

DISCUSSION apparently normal fashion. The morphologic evi-

The studies presented in this report document the dence for degranulation from light microscopy isability of leukocytes from a patient with CGDto supported by the electron microscopic studies.undergo degranulation and vacuole formation in The most extensive studies of the metabolism of

1760 E. Kauder, L. L. Kahle, H. Moreno, and J. C. Partin

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FIGURE 6 Comparison of 02 consumption by leukocytesof the patient and those of a normal subject. Note that02 content decreased after the addition of polystyrenelatex particles to normal leukocytes, indicating consump-tion of 02. This did not occur with the patient's leuko-cytes.

PMNfrom patients with CGDare those of Holmesand associates (5). They have reported that phag-ocytosis fails to stimulate respiration, hydrogenperoxide production, and HMSactivity. Relatingthese observations with those previously publishedby Quie and coworkers Holmes et al. have hypothe-sized that the metabolic abnormalities are tied tothe failure of degranulation and of intracellulardeath of bacteria.

Wehave demonstrated a lack of stimulation ofrespiration in the PMNof our patient and a failureof his leukocytes to reduce NBT during phagocy-tosis. These metabolic abnormalities, in conjunc-tion with the typical clinical course, pathologicalfindings, and suggestion of sex-linked inheritanceof his disease, make it most likely that he is suf-fering the same disease as those patients reportedby the investigators cited above. We think itreasonable to suggest that the metabolic abnor-mality of leukocytes in patients with CGD is notdependent upon a failure of degranulation by thesecells. Support for this suggestion comes from thedata of Zatti and coworkers (7) who have notedthat stimulation of HMSactivity as measured by

4CO2 production from a glucose-1-14C precursoris temporally unrelated to and clearly precedes theevent of degranulation in the test leukocytes.

An alternative hypothesis is that our patient hasa different cellular defect than those previously re-ported and that any metabolic error in the leuko-cyte leading to defective intracellular killing ofbacteria can produce the same clinical syndromeof CGD. Failure to kill intracellular bacteria couldresult from defective bactericidal substances withinthe cell or from a failure to deliver the bactericidalmaterial from its lysosomal package in the cyto-plasmic granule to the membrane-bound vacuolewhich is the intracellular environment of the in-gested bacteria. In our current state of knowledgewe feel, however, that the more tenable hypothesisis one which accepts that the event of degranula-tion is not causally related to the metabolic dys-function as described by Holmes and coworkers(5), by Baehner and Nathan (6), and as seen inour patient.

Skarnes and Watson (18) have extracted a pro-tein from the nucleus of the PMNwhich mani-fested selective bactericidal activity against Gram-positive organisms. Zeya and Spitznagel (19)have recently extracted a cationic protein fromthe PMNlysosomes which was equally bacterici-dal against Gram-positive and Gram-negative or-ganisms. It is therefore possible that specific pro-tein defects might result in susceptibility to onlycertain strains of bacteria. The definitive answermust await further studies.

ACKNOWLEDGMENTS

We are grateful to Miss Ann Damon, Miss VirginiaFisher, Mrs. Carol Whidden, and Mrs. Ruth Wright fortechnical assistance, and to Mrs. India Oldham forpreparation of the manuscript. We are indebted to Dr.George Benzing III for. studies of oxygen consumption.

This investigation was supported by grants f rom theU. S. Public Health Service (HD 02314, CA 04826, andFR 00123). Dr. Kauder is a recipient of a special fellow-ship award (IF 3 AM-34943) from the Institute of Ar-thritis and Metabolic Diseases of the U. S. Public HealthService. Dr. Partin is a recipient of a special fellowshipaward from Child Health and Human Development(HD 02443-OlAl).

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9. Maaloe, 0. 1946. On the Relation between Alexin andOpsonin. Ejnar Munksgaard, Copenhagen.

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12. Severinghaus, J. W. 1958. Oxyhemoglobin dissociationcurve correction for temperature and pH variation inhuman blood. J. Appl. Physiol. 12: 485.

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15. Windhorst, D. B., B. Holmes, and R. A. Good. 1967.A newly defined X-linked trait in man with demon-stration of the Lyon effect in carrier females. Lancet.1: 737.

16. Graham, G. S. 1918. Benzidine as a peroxidase reagentfor blood smears and tissues. J. Mlcd. Res. 39: 15.

17. Ackerman, G. A. 1962. Substituted naphthol as phos-phate derivatives for localization of leukocyte alkalinephosphatase activity. Lab. Invest. 11: 563.

18. Skarnes, R. C., and D. W. Watson. 1956. Characteri-zation of leukin: an antibacterial factor from leuko-cytes active against Gram-positive pathogens. J. Exptl.Med. 104: 829.

19. Zeya, H. I., J. K. Spitznagel. 1966. II. Composition,properties, and mechanism of antibacterial action.J. Bact. 91: 755.

1762 E. Kauder, L. L. Kahle, H. Moreno, and J. C. Partin