Opsonic Activity in the Newborn: Role of Properdin

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REFERENCES

1. Gregory, C. A., Kitterman,

J.

A., Phibbs, R. H.,

Tooley, W. H., and Hamilton, W. K.: Treat-ment of the idiopathic respiratory distress syndrome with continuous positive airway pressure. New Eng. J. Med., 284: 1333, 1971. 2. Chernick, V., and Vidyasagar, D.: Continuous

negative chest wall pressure in hyaline mem-brane disease: One year experience.

PinxT-RICS, 49:753, 1972.

3. Fanaroff, A. A., Cha, C. C., Sosa, R., Crumrine, R. S., and Klaus, M. H.: A controlled trial of

continuous negative external pressure in the

treatment of severe respiratory distress syn-drome.

J.

Pediat., to be published.

4. Barrie, H.: Simple method of applying continu-ous positive airway pressure in respiratory distress syndrome. Lancet, 1 :776, 1972. 5. Harris, T. R. : Continuous positive airway

pres-sure applied by face mask. Pediat. Res., 6: 410, 1972,.

6. Arp, L.

J.,

Dillon, R. E., Humphries, T.

J.,

and

Pierce, D. E.: A new approach to ventilatory support of infants with respiratory distress

syndrome. Anesth. Anaig., 48:506, 1969. 7. Agostino, R., Orzalesi, M., NOdari, S.,

Mendi-cliii, M., Conca, L., Savignoni, P. C.,

Picece-Bucci, S., Calliumi, C., and Bucci, C. : Con-tinuous positive airway pressure (CPAP) by

nasal canula in the respiratory distress

syn-drome (RDS) of the newborn. Pediat. Res., 7:50, 1973.

Opsonic

Activity

in the Newborn:

Role of Properdin

The

known susceptibility of some newborn

infants to bacterial infections has been attrib-uted to a deficiency of serum opsonins.13 In

this report, we describe measurements of

op-sonic activity of cord serum for particles shown to depend on the properdin system for opsoni-zation. Most cord sera tested had nearly nor-ma! activity relative to adult sera. Markedly defective activity in 15% of the sera correlated

with

subnormal levels of factor B of

the

pro-perdin

system,

a fl-globulin

of serum

also

des-ignated

GBG

and

C3PA.

The heat-labile opsonic activity of normal

serum involves

the

fixation of the third corn-ponent of complement (C3) to

the

surface of

microorganisms. This fixation may occur by

means of sequential reactions involving

anti-body and the first three components of the

classical complement pathway (Cl, C4, and

C2)’ or else by an incompletely defined

alter-nate pathway, the properdin system.5 Antibody is not essential for opsonization by the

proper-din ecan’6 A number of serum proteins

of potential importance for opsonic activity are

relatively deficient or absent from cord serum. 1gM is in decreased concentration in most cord

sera,#{176}

and

transplacentally derived IgG

anti-body to gram-negative endotoxin is only

oc-casionally found.8 Cl, C4, C2, and C3 are

found in cord sera in approximately two-thirds of the levels observed in

normal

adult sera.9 .#{176}

A component of the properdin system,

glycine-rich beta glycoprotein (GBG)

,

(also

desig-nated the C3 proacfivator or factor B) is do-creased compared

with

normal

adult sera and, in some cord sera, is markedly diminished.”

MATERIALS AND METHODS

Sera were separated from the cord blood of neonates within eight hours of collection and stored at -70 C. Sera were also obtained from 20 normal adults, pooied and stored as

do-scribed above. For preparation of

periph-eral leukocytes, blood was

collected

from nor-mal humans in 0.2 volumes of ACD

anti-coagulant. Erythrocytes were sedimented by standing for 45 minutes at room temperature

after addition of 0.3 volumes of 6% dextran

(500,000 molecular weight) in 0.15 M sodium

chloride. Residual erythrocytes in the

super-natant fluid were lysed by addition of 3

vol-umes of 0.87% ammonium chloride. The

leuko-cytes were washed twice

with

50 ml of 0.15 M

sodium chloride at 4 C and suspended in

Krebs-Ringer phosphate medium, pH 7.4, at a final concentration of 20,000 to 40,000 cells per mm.

Paraffin oil particles containing Oil Red 0 and stabilized with Escherichia coli

026:B6

lipopolysaccharide were prepared by sonica-tion of paraffin oli-Oil Red 0 in a lipopoly-saccharide suspension as previously described.’ These particles were opsonized by adding 0.2

ml of cord or pooled adult serum to 0.2 ml of particle suspension and incubating at 37 C for

15 minutes.

To assay the rate of ingestion of opsonized

particles, 0.8 ml of leukocyte suspensions were

(2)

100

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Is

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75.

j

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5.

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25

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0 #{149}

50 100 150 200 250 300 350

I:: I00

75

50

25

-

,:‘I

.

:

.

#{149}.#{149}#{149}

.1 #{149}#{149}

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.

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500 1000 1500

135/174

EXPERIENCE

AND

REASON-BRIEFLY

RECORDED

L

I:::

086

(mg/LI

Fic. 1. Opsonic activity of cord sera as a function of C3 concentration. The

opsonic activities of the cord sera are expressed as percent of opsonic ac-tivity of pooled normal adult serum which had a C3 concentration of 1230 mg per liter. The vertical line indicates the lower range of normal adult

C3 concentration.

four

minutes

and

were

then

terminated

by the

addition of 6 ml of ice-cold 0.5 M sodium chloride

1 mM

N-ethyl

maleimide

to the

tubes.

Uningested particles

were

removed

from

the

cells

by

centrifugation

twice

at

150X

g, and

the

washed cell pellets were then extracted

with p-dioxane. The initial rate of uptake of

paraffin oil was calculated from the optical density of Oil Red 0 in the dioxane extracts at

425 nm as previously described.” Because of

variability of ingestion rates by different

leuko-cyte preparations, the results were expressed

C3

(mg/L

I

Fic. 2. Opsonic activity of cord sera as a function of GBG, concentration. The opsonic activities of the cord sera are expressed as percent of opsonic activity of pooled normal adult serum which had a GBG concentration of 330 mg per liter. The vertical line indicates the lower range of normal

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138/175

as the percent uptake relative to the ingestion rate of particles opsonized

with

pooled

adult

serum.

The concentrations of C3 and GBG in sera

were

quantitated

by electroimrnunoassay.1’

Levels

of IgG,

IgA,

and

1gM

were

determined

by

quantitative

immunoprecipitation.”

RESULTS

Of 40 cord

sera

tested,

34,

or 85%, had

op-sonic activity between 70% and 110% of pooled

adult serum.

Six

of the cord sera had activity

between 43% and 65% of adult serum. These

cord sera had <5 rng/100 ml of 1gM or IgA.

The levels of IgG were within the normal range (650 to 1,610 rng/100 ml) .

Three-fourths of the cord sera had C3 levels below the lower range of normal (100 mg/100 ml), but there was no correlation between opsonic activity

and

the C3 concentration (Fig. 1). Although all of the cord sera had lower GBG levels

than

the pooled adult serum, only six of

the sera had GBG concentrations below the lower limit of normal (12 mg/100 ml) . These six sera had the lowest opsonic activity (Fig.

2) . There was no correlation between

gesta-tional age or birth weight and serum opsonic

activity.

DIscussIoN

Paraffin oil droplets coated

with

E. coli

lipo-polysaccharide are ingested slowly by human

peripheral blood granulocytes and monocytes

unless they

are

first

opsonized. Therefore, the initial rate of ingestion of the particles by leu-kocytes reflects the opsonic activity of serum used to treat the particles, and the opsonic activity has been shown to depend on C3 and

the properdin system.’ Since newborns tend to have lower serum C3 concentrations than nor-mal adults,” the moderate deficiency in opsonic activity of most cord sera relative to pooled

adult serum could be explained partially on

this basis. However, there is considerable

van-tion in opsonic activity of adult sena.1’

There-fore, the mild opsonic deficiency in cord sena

demonstrated in comparison to pooled adult serum may not be of great significance. How-ever, a small fraction of cord sena had

marked-ly deficient opsonic activity. These sena did not differ significantly from the other cord sera with respect to

C3

or

immunoglobulin

concentra-tions. However, they all had subnormal concen-trations of GBG, which is an essential

compo-nent of the propendin system. Addition of

purl-fled

GBG to one of these sena has previously

been shown to increase its opsonic activity.6

Dysfunction of the properdin system results in hypersusceptibility to bacterial infections,’7

but humans and animals deficient in C2 or

C4 do not have

this

susceptibility.”

We

infer,

therefore, that the deficient opsonic activity of some of the cord sera is due to hypofunction

of the alternate complement pathway and that

this impairment may render certain neonates unduly susceptible to infection.

Suiniy

Relative to pooled adult serum, most cord sera have normal or slightly diminished opsonic activity. Fifteen percent of the cord sera had

markedly impaired opsonic power. This activ-ity did not correlate

with

immunoglobulin or

C3 levels but was associated

with

significantly

subnormal concentrations of GBG, a

compo-nent of the properdin system. The deficient opsonic activity of some cord serum is thus due to hypofunction of the alternate comple-ment pathway.

THOMAS

P.

STOSSEL,

CHESTER A. ALPER,

FRED S. ROSEN

Divisions of Hematology and

Immunology,

Department of Medicine,

Children’s Hospital Medical Center, Center for Blood Research,

Department of Pediatrics, Harvard Medical School

Boston, Mcsachusetts

02115

Supported by USPHS grants AM-13855,

A!-05877, and NHLI-72292B.

T.P.S. is an Established Investigator of the

American Heart Association.

REFERENCES

1. Miller, M. E. : Phagocytosis in the newborn infant: Humoral and cellular factors. J. Pediat., 74:255, 1969.

2. Forman, M. L., and Stiehm, E. R. : Impaired

opsonic activity but normal phagocytosis in low birth weight infants. New Eng. J. Med.,

281:926, 1969.

3. Dossett, J. H., Williams, R. C., and Quie, P. C.: Studies on interaction of bacteria, serum factors, and polymorphonuclear leukocytes

in mothers and newborns. Psusmics, 44:

49, 1969.

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EXPERIENCE AND

REASON-BRIEFLY

RECORDED

137/176

of bacterial phagocytosis by serum. The role of complement and two co-factors. J. Exp. Med., 129:1275, 1969.

5. Stossel, T. P., Alper, C. A., and Rosen, F. S.: Phagocytosis of paraffin oil emulsified with

bacterial lipopolysacchanide. J. Exp. Med.,

137:690, 1973.

6. Jasin, H. E. : Human heat-labile opsonins: Evidence for their mediation via the alter-nate pathway of complement activation. J. Immun., 109:26, 1972.

7. Gitlin, D., Rosen, F. S., and Michael, J. G.:

Transient 19S gamma-globulin deficiency in the newborn and its significance.

PnimT-RICS, 31:197, 1963.

8. Gupta, J. D., and Reed, C. E.: Natural

anti-bodies to Salmonella enteritidis endotoxin in maternal and cord sera and in sera of pa-tients with immunologic deficiency diseases. mt. Arch. Allerg., 31 :324, 1968.

9. Fishel, C. W., and Pearlman, D. S.: Comple-ment components of paired mother-cord

sera. Proc. Soc. Exp. Biol. Med., 107:695,

1961.

10. Propp, R. P., and Alper, C. A.: C3 synthesis in the human fetus and lack of transplacen-tal passage. Science, 162:672, 1968. 11. G#{246}tze,0., and Muller-Eberhard, H. J.: The

C3 activator system : An alternate pathway of complement activation. J. Exp. Med., 134:902, 1971.

12. Alper, C. A., Boenisch, T., and Watson, L.:

Genetic polymorphism in human glycine-rich beta-glycoprotein. J. Exp. Med., 135:

68, 1972.

13. Stossel, T. P., Mason, R. J., Hartwig, J.,

and

Vaughan, M. : Quantitative studies of phag-ocytosis by polymorphonuclear leukocytes. Use of emulsions to measure the initial rate of phagocytosis. J. Clin. Invest., 51:614,

1972.

14. Laurel, C. B.: Quantitative estimation of pro-teins by electrophoresis in agarose gel

con-taming antibodies. Anal. Biochem., 15:45,

1966.

15. Ritchie, R. F., Alper, C. A., and Graves, J.: Automated quantitation of proteins in serum and other biological fluids. Amer. J. Cliii.

Path., in press.

16. Stossel, T. P. : Evaluation of opsonic and leukocyte function with a spectrophoto-metric test in patients with infection and

with phagocytic disorders. Blood, to be

published.

17. Alper, C. A., Abrainson, N., Johnston, R. B., Jr., Jandl, J. H., and Rosen, F. S. : Increased

susceptibility to infection associated with abnormalities of complement-mediated func-tions and of the third component of comple-ment (C3). New Eng. J. Med., 282:349, 1969.

18. Alper, C. A., and Rosen, F. S.: Genetic aspects

of the complement system. Adv. Immun., 14:251, 1971.

The Demonstration

of Insulinopenia

in Familial

Dysautonomia

Familial dysautonomia (Riley-Day syn-drome) is a congenital disease occurring

al-most entirely in persons of Jewish ancestry. It is characterized by absent lacrimation, ex-cessive perspiration, impaired temperature

control, labile blood pressure, poor swallowing,

excessive salivation, frequent vomiting,

hype-active deep tendon reflexes, indifference to pain, absent fungiform papillae on the tongue,

and mental and motor retardation.

Riley’ found a marked retardation in both height and weight of all 27 patients studied

with

dysautonomia. Almost all patients were

be-low the

10th

percentile,

and

many

were

below

the 3rd percentile for both parameters. He also concluded that height and weight at the 50th percentile for age is a factor against the diag-nosis.

Basic studies of the carbohydrate

metabo-lism and sequential changes in serum insulin

and growth hormone (GH) in subjects with fa-milial dysautonomia have not been previously reported. It is the objective of this study to determine whether abnormalities in any of these variables, which might be responsible for

growth failure, are demonstrable.

MATERIALS AND METHODS

Nine children with dysautonomia (ages 3% to

17 years) and seven healthy control children (ages 4 to 17% years) were studied. All

chil-dren were of Jewish ancestry and were ambula-tory at the time of testing. For three days prior to testing, the children were maintained on a

diet of approximately 50% of the calories as carbohydrate. A rapid intravenous0 glucose tol-erance test (IVGTT) was performed on all children in the morning and after a 12-hour fast,

by a modification of the method of Soeldner.2

Glucose was administered intravenously as a 25% solution in a dosage of

0.5

gm per kilo-gram body weight. This intravenous infusion

was completed within 2 to 4 minutes, and the clock was started at that time.

Capillary blood was collected and analyzed

0 Oral administration of glucose was attempted

with the first three dysautonomic children; all vom-ited it. Since vomiting is so characteristic of the syndrome, it was apparent that only an intravenous

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1973;52;134

Pediatrics

Thomas P. Stossel, Chester A. Alper and Fred S. Rosen

Opsonic Activity in the Newborn: Role of Properdin

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