the septic burned patient

The Septic Burned Patient

A Model for Studying the Role of Complement and Immunoglobulins inOpsonization of Opportunist Micro-organisms

ANN B. BJORNSON, PH.D., WILLIAM A. ALTEMEIER, M.D., H. STEPHEN BJORNSON, M.D., PH.D.

Studies were performed to determine the effects of septicemiaon complement levels and activities and opsonic function inseptic and nonseptic burned patients. None of the nonsepticburned patients had consumption of classical pathway activityduring their clinical course. Patients who did not survivesepticemia had consumption of all of the classical complementcomponents (C1-C5) prior to and during their septic episodes.Patients who survived septicemia had multiple patterns ofclassical complement pathway consumption. In these patients,classical pathway activity was restored to normal following thelast positive blood culture. Alternative complement pathwayconsumption was demonstrated in only one of the septic burnedpatients, as evidenced by decreased factor B and C3b INAlevels and decreased C3 and C5 conversion in sera treated with10 mM ethylene glycol tetraacetic acid and 10 mM MgCl2(MgEGTA) and in untreated sera. In all of the other septic pa-tients and in the nonseptic patients, reduction in C3 and CSconversion in MgEGTA sera and untreated sera was not asso-ciated with decrease in factor B or C3b INA. Reduction incomplement levels and activities did not reduce the ability ofthe patients' sera to promote phagocytosis and intracellularkilling of their infecting micro-organisms by normal humanperipheral polymorphonuclear leukocytes. The results indicatethat measurement of classical pathway activity in burned pa-tients can be used as a diagnostic tool for predicting the severityof septic episodes and for monitoring recovery. In addition,the observation that complement consumption did not reducethe opsonic capacity of the patients' sera for their infectingmicro-organisms suggests that current concepts regarding therole of immunoglobulins and complement in opsonization ofopportunist micro-organisms require re-evaluation.

IN OUR PREVIOUS PRELIMINARY STUDIES, reductionin the immunochemical levels and functional ac-

tivities of components of the classical and alternativecomplement pathways was associated with septicemiain two burned patients.1 Studies on a group of five addi-

Reprint requests: A. Bjornson, Ph.D., Division of InfectiousDisease, Department of Medicine, University of Cincinnati Collegeof Medicine, 231 Bethesda Ave., Cincinnati, Ohio 45267.Supported by the U.S. Army Medical Research and Development

Command under Contract #DAMD 17-76-C-6023.Submitted for publication: October 20, 1978.

From the Departments of Medicine and Surgery,University of Cincinnati College of Medicine,

Cincinnati, Ohio

tional septic burned patients showed that reduction inthe classical complement pathway occurred prior toand during septic episodes in three of the patients.2 Inthe other two patients, decreased classical pathway ac-tivity was demonstrated prior to the development ofsepticemia, but not consistently during septic episodes.Reduction in the functional activity of the alternativecomplement pathway was not found to be associatedwith septicemia in any of the five burned patients in thisstudy group. Reduction in classical and alternativecomplement pathway activity did not reduce the op-sonic capacity of the patients' sera for the specificmicro-organisms causing infection. In only one patient,consumption of components of the classical pathwayoccurring during septicemia decreased the opsoniccapacity of the patient's sera for her own infectingmicro-organism, an isolate of Escherichia coli; serafrom the same patient which could not opsonize E. coliopsonized her infecting strain of Staphylococcusaureus normally.The objectives of the present investigation were as

follows; a) Determine if the classical complement path-way was consumed preferentially during septicemia inburned patients, or if alternative complement pathwayconsumption could occur as well. b) Determine if theclassical pathway was always reduced during the initialpostburn period in patients who later developed sep-ticemia. c) Determine if reduction in serum opsonic ac-tivity occurred for certain infecting bacteria and not forothers, as was observed in our previously studied groupof septic patients. d) Evaluate the effects of the ad-ministration ofblood products on the observed changesin humoral factors in septic and nonseptic burned pa-

0003-4932/79/0400/0515 $01.15 © J. B. Lippincott Company

515

BJORNSON, ALTEMEIER AND BJORNSON

tients and on the outcome of the infections in the sep-tic patients. e) Provide a detailed study of the func-tional integrity of the alternative complement pathwayin septic and nonseptic burned patients; and f) Provideinformation about the immunochemical concentrationsof classical pathway components in nonseptic burnedpatients, since in our previous studies only functionalactivity of the classical pathway was measured in thispatient population.

Materials and Methods

Patients

Eleven patients with severe thermal injury were fol-lowed during nine weeks postburn. Patients who ap-peared to be at the greatest possible risk of infection be-cause of burn size or age or both were selected for thestudy. Serum samples were obtained from the patientsas soon after the injury as possible and then at weeklyintervals. In those patients who developed septicemia,serum samples were also obtained two additional timesper week until blood cultures became negative. Bloodcultures were drawn on all patients at least one time perweek by our staff, and additional blood cultures weredrawn at the discretion of the attending physicians.This procedure was adopted for the purpose of docu-menting negative as well as positive cultures obtainedon the patients. Septicemia was documented by clinicalfindings and positive blood cultures. The clinical cri-teria used for the diagnosis of septicemia were a) chillsand fever, b) tachycardia, c) hypotension, and c) dis-orientation.

Handling ofBlood Samples

The blood samples for serologic studies were allowedto clot for one hour at room temperature, then for onehour at 40, and were centrifuged at 5,000 x g. The serawere removed, dialyzed against 0.01 M phosphate buf-fered saline, pH 7.3, containing 1.5 x 10-4 M CaCl2and 5.0 x 10-4 M MgCl2 overnight, divided intoaliquots, and frozen at -70°. Dialysis of the sera wasperformed to remove antibiotics and to standardize theconcentration of magnesium and calcium ions in thesera. Normal human sera were collected from 20healthy adult donors, dialyzed in a similar fashion, andstored individually at -70°. In some experiments,normal or burn sera were treated with 10 mM ethyleneglycol tetraacetic acid and 10 mM MgCl2 (MgEGTA).3Total hemolytic complement was not detected in seratreated with MgEGTA, indicating that classical path-way activity was blocked in the sera.Blood samples (8ml) from normal adult volunteers

for preparation of polymorphonuclear leukocytes

(PMNs) were collected in heparinized (10 units/ml)plastic tubes containing 2ml of 0.6% dextran in saline,pH 7.0. After sedimentation of the erythrocytes atroom temperature for one hour, the leukocyte-richplasma was removed. The leukocytes were washedtwice in Hank's Balanced Salt Solution without phenolred (Microbiological Associates, Inc., Bethesda, Md.)containing 0.1% gelatin (HBG) and were resuspendedin HBG to contain 1.0 x 107 PMNs/ml.

Growth ofthe Micro-organismsfor the Opsonic Assays

The micro-organisms isolated from the patients weremaintained in brain heart infusion broth and werefrozen at -70°. Prior to each experiment, a frozen cul-ture was thawed, plated on brain heart infusion agar,and the plate was incubated at 370 overnight. A singlecolony was then transferred to a tube of brain heart in-fusion broth which was incubated at 37° overnight.The micro-organisms were washed twice and resus-pended in HBG to a concentration of 1.0 x 108 cells/ml.

Measurement of Serum Opsonic Activity

A minor modification of the method of Hirsch andStrauss5 was used for measuring PMN bactericidal ac-tivity. To summarize the methodology, 1.0 x 106micro-organisms and 5.0 x 106 normal PMNs were in-cubated with the test serum and HBG in a final volumeof lml. HBG was substituted for the serum or leuko-cytes in the controls. Duplicate reaction mixture andcontrol tubes were rotated end over end at 370. At 0time and after one and two hours of incubation, thenumber of bacteria surviving in the controls and reac-tion mixtures was determined by plating three ten-folddilutions of the samples in distilled water on brain heartinfusion agar and colony counting after overnight in-cubation. The per cent of micro-organisms phago-cytosed and killed intracellularly was determined bythe formula (a - b/a) x 100 where a was the number ofbacteria surviving in the control lacking leukocytes andb was the number of bacteria surviving in the corre-sponding reaction mixture containing leukocytes. Theper cent of serum selected for the assays was deter-mined for each bacterial strain in a preliminary experi-ment and was the minimal concentration of poolednormal human serum which promoted maximal phago-cytosis and killing of the strain by normal PMNs dur-ing the shortest incubation period.

Radial Immunodiffusion Assays

Single radial immunodiffusion was performed by aminor modification of the method of Mancini et al.7 Forassaying concentration of B antigen of C3, factor B,

516 Ann. Surg. * April 1979

SEPTIC BURNED PATIENT

C3b INA, Clq, C4, C2, and C5, agarose (1%) dis-solved in veronal buffer (,u = 0.05, pH = 8.6) contain-ing 0.04 M EDTA was used. For assaying properdinconcentration, agarose (0.6%) dissolved in 0.01 Mphosphate buffered saline, pH 7.0, containing 0.01 MEDTA was used. An appropriate dilution of antiserumin agarose was poured into plastic plates which werestored at 40 overnight. For assays of factor B, C4, andC3, thirty 2mm diameter wells were made on eachplate, and 3,u1 of serum were delivered into each well.For assays of Clq, C2, C5, C3b INA, and properdinconcentration, 20 4mm diameter wells were made oneach plate, and 10/lO of serum were delivered into eachwell. The unknowns and serial dilutions of the standardserum for the protein being measured were placed in thewells, and diffusion was carried out at room tempera-ture for 24 hours (Clq-C5, factor B, and C3b INA) orat 370 for 48 hours (properdin). All determinations wereperformed in duplicate.*

Purified Proteins and Antisera

Human factor B was purified according to themethod of Gotze and Muller-Eberhard.4 Properdin wasisolated by the method of Pensky et al.,10 except that aeuglobulin fraction of human serum rather than serumreacted with zymosan was used as the starting material.By polyacrylamide gel electrophoresis, the final prep-arations of properdin and factor B were found to becontaminated only with trace amounts of gammaglobulin. Human C3b INA, C4, C2, and C3 werepurchased from Cordis Laboratories, Miami, FL.

Antisera to properdin, factor B, C3b INA, C4, andC3 were raised in goats by multiple subcutaneous in-jections of the respective partially purified protein insaline containing 0.01 M EDTA, pH 7.0 (EDTA-saline) incorporated in Freund's complete adjuvant.Antiserum to C2 was raised in goats by multiple intra-venous injections of human C2 in EDTA-saline. Anti-serum to B antigen of C3 was prepared by absorbingantiserum to C3 with the minimal amount of aged nor-mal human serum which fully removed antibodies tothe A and D antigens.14 The antisera to factor B andC2 were absorbed with heated normal serum (500, 30min). The antiserum to properdin was absorbed withproperdin depleted serum'3 and the antiserum to C3bINA was absorbed with normal human serum heatedat 560 for two hours. All of the procedures removedantibodies to contaminating proteins. Antisera to

human C4 did not contain contaminating antibodies.Each antiserum in agarose yielded single lines againstnormal human serum in immunoelectrophoresis andcomplete identity in immunodiffusion with referencegoat antiserum to each respective protein.t

Hemolytic Assays

Total hemolytic complement was titrated by themethod of Kabat and Mayer.8 Hemolytic C3 and C5were titrated by the method of Nelson et al.9 using avolumetric modification. EAClgp4hu and purifiedhuman C2, C6, C7, C8, C9, and C3 or C5 were used;the cellular intermediates and purified componentswere purchased from Cordis Laboratories. The diluentused for the experiments consisted of 2.5% glucose inveronal buffer8 containing 0.0003 M CaCl2, 0.001 MMgCl2, and 0.1% gelatin. All of the results were origi-nally expressed in CH50 units/ml; values for hemolyticC3 and C5 were converted to per cent of normal toallow comparisons between determinations to be made.

Measurement of C3 and CS Conversion

Inulin (100 mg/ml) and cobra venom factor (CoVF)(500 units/ml) in saline were added to sera in the pro-portion of 1Oul of activating substance to lOO1l ofserum, and the mixture was incubated for one hour at370. Sera treated with saline served as controls for theexperiments. Concentration of B antigen of C3 wasdetermined by radial immunodiffusion before and afterincubation with the activating substance, and the dif-ference in concentration was taken as a measure of C3conversion." C3 conversion was also determined bymeasuring residual hemolytic C3 before and after in-cubation with the activating substance. Conversion ofC5 was determined by measuring residual hemolyticC5 before and after incubation with the activating sub-stance. The results of the assays were expressed in percent of normal.

Results

Seven of the 11 patients had positive blood culturesduring their clinical course. Clinical signs of septicemiawere documented in all of these patients, when bloodcultures were positive. Clinical characteristics of thisgroup of patients and information regarding blood cul-tures are given in Table 1. The other four patients hadnegative blood cultures. Clinical characteristics of this

t The reference antisera to factor B, C3b INA, properdin, C4, C2,and B antigen of C3 were generously provided by Dr. C. West. Rab-bit antiserum to human Clq and C5 were purchased from BehringDiagnostics, Somerville, NJ.

* Normal reference sera to B antigen of C3, C4, C5, and factor Bwere kindly provided by Dr. C. West, Childrens Hospital ResearchFoundation, Cincinnati, Oh., and for properdin, C3b INA, Clq, andC2 was a pool of 25 normal sera.

Vol. 189.o No. 4 517

Ann. Surg. * April 1979BJORNSON, ALTEMEIER AND BJORNSON

TABLE 1. Clinical Characteristics of the Septic Burned Patients

Body Surface InjuredtBlood Culturest

Third InfectingPatient Total Degree Micro- Positive NegativeNo. Age Sex* Per Cent Per Cent organisms Day Day

I 4 F 48 48 S. aureus 17,24,28,31,35, 18,45,5236,37,38,40,42

II 3 F 77 70 E. coli 10 4,30,41S. aureus 17,18,20P. aeruginosa 23,29

III§ 55 F 52 17 P. aeruginosa 17,18,19,20 5,9,12

IV 11 M 51 13 S. aureus 13,31,34 17,24,38,45

V§ 14 F 38 35 S. aureus 37,39 19,23,25,32

VI§ 9 F 67 67 S. aureus 2 1E. coli 4K. pneumoniae 4,7,11,14,18,21,23C. freundii 4S. faecalis 4,5,7,9P. aeruginosa 4

VII 37 M 59 54 S. aureus 15,24S. faecalis 15 5,13,17,20,

21,29

* M = male; F = female. tAll patients had flame burn injuries.tNumbers indicate the number of days following the injury that

group of burned patients are shown in Table 2. Datafrom the nonseptic patients will be presented first, fol-lowed by the data on the septic patients, to allow ap-

propriate comparisons between the two patient popula-tions to be made.

Total hemolytic complement (CH50) fell within thenormal range in all of the nonseptic burned patients forthe duration of the study, despite decreased immuno-chemical levels of Clq and C2 in the sera of Patient 2during ten days postburn (Fig. 1). The immuno-chemical level of C4 was found to be normal in all of thepatients for the duration of the study. The immuno-chemical level of B antigen of C3 was also found to benormal in all of the patients for the entire study period(Fig. 2). However, the immunochemical level of C5 fellbelow two standard deviations from the normal mean inPatients 1 and 2 during 30 days postburn. Functionalactivities of C3 and C5 were also measured in thesera of the four patients by standard hemolytic meth-ods. The normal ranges for the assays were so broadthat only one standard deviation from the normal meancould be presented in the figure. All of the patients hadnormal or elevated C3 and C5 as measured by thesemethods.

Properdin in one of the four patients was decreasedbelow two standard deviations from the normal mean

during 20 days postburn, and in the other three patients,fell below the normal mean value until the fourty-firstto fiftieth postburn day period (Fig. 3). C3b INA and

positive or negative blood cultures were obtained. §These patientsdied of septic shock on Day 21 (#3), Day 41 (#5), and Day 23 (#6).

factor B levels in all of the patients were normal orelevated during the entire study period.

Utilizing reduction in the B antigenic determinant ofC3 by radial immunodiffusion as a method for meas-uring C3 conversion, Patient 1 had reduced C3 con-version by inulin during five days postburn and bycobra venom factor (CoVF) during ten days postburn;C3 conversion by both activating substances in thispatient's sera was restored to normal thereafter. Pa-tient 2 had normal C3 conversion by inulin and CoVFuntil the twenty-first to thirtieth postburn period whichwas subsequently restored to normal. Patients 3 and 4had normal C3 conversion by both activating sub-stances for the duration of the study using thismethod. The hemolytic assay for measuring C3 con-version by inulin yielded different results. C3 conver-

TABLE 2. Clinical Characteristics of the Non-SepticBurned Patients

Body Surface Injuredt

ThirdPatient Total DegreeNo. Age Sex* Per Cent Per Cent

1 59 M 62 02 14 M 43 333 12 M 40 04 15 M 49 45

* M = male. tAll patients had flame burn injuries.

518

Vol. 189 No. 4

150

100

FIG. 1. Immunochemicallevels and functional ac-

tivities of components ofthe classical complementpathway in the sera of fournonseptic burned patientsduring 50 days postburn.The shaded areas representthe variation in 20 in-dividual normal sera (mean± 2 S.D.). The Arabicnumerals following thelines represent patientnumbers. Refer to Table 2for the clinical characteris-tics of the patients. Thepoints represent meanvalues of duplicate deter-minations.

501


CH50

*a +2S.D.

- eMEAN

-2S.D5

I I I

)1I-5 6-10 11-20 21-30 31-40 41-SO

Clq150 r

0

z

LA-

O05C

4

/

ME

..a -24

0 IS 6 - 21-0 -

0 1-5 6-10 11-20 21-30 31-40 41-50

tS.D

EAN

SD.

ar0z

U.0

519150 r

100

E

C4

4

/L- - +2S.D.

-~~~~~7 -------s0 MEAN

0 1-5 6-10 11-20 21-30 31-40 41-!

-2S.D.

50o

C2

loo - .' 400~~~~~_!~~ +2SD

50 Mommo\\\g m - DEAN50 -2SD

0 1-5 6-10 11-20 21-30 31-40 41-S0

DAYS POSTBURN

FIG. 2. Immunochemical and hemolyticmeasurements of C3 and C5 in the seraof four nonseptic burned patients during50 days postburn. The shaded areas

represent the variation in 20 individualnormal sera (mean 1 S.D. for hemolyticdeterminations and mean 2 S.D. for im-munochemical determinations). TheArabic numerals following the linesrepresent patient numbers. Refer toTable 2 for the clinical characteristics ofthe patients. The points represent meanvalues of duplicate determinations.

0z

Li.

0

$N

100 _ C3 (lmmunodwmicol)90 -

80 _

ffi 60 -

30 MAN

ae

10 _ I_Ig oI IL0 1-5 6-90 1-20 21-30 3-40 41-50 0

DAYS POSTBURN

r- CS (Hemolytic)

2

+.IS.D

MEAN

-'sn

0 I-5 6-10 11-20 21-30 31-40 41-50

MF.JRMS.x.x.x.\ __

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Factor B 200

I D+2SD

MEAN

0) -5 6-10 11-20 21-303\1m-40 41-

0 1-5 6-10 1 1-20 21-30 31-40 41-50

-J4

0zL-

0I

SrD

C3b INA

0 1-5 6-10 11-20 21-30 31-40 41-!

+2SD.

MEAN FIG. 3. Immunochemicallevels of factor B, pro-

-2S.D perdin, and C3b inactivator(C3b INA) in the sera offour nonseptic burned pa-tients during 50 days post-burn. The shaded areasrepresent the variation in 20

50 individual normal sera(mean + 2 S.D.). TheArabic numerals followingthe lines represent patientnumbers. Refer to Table 2for the clinical characteris-tics of the patients. Thepoints represent meanvalues of duplicate deter-minations.

0 1-5 6-10 11-20 21-30 31-40 41-50

DAYS POSTBURN

sion by inulin was reduced in Patient 1 during 30 dayspostburn and was subsequently normalized. C3 con-

version by inulin in Patients 2, 3, and 4 was consistentlynormal for the duration of the study using this method.Treatment of the burn sera with MgEGTA, whichblocked classical pathway activity, did not substan-tially reduce C3 conversion by inulin in the sera. C5conversion was normal for the entire study period ex-

cept in Patient 1 during the thirty-first to fortieth post-burn day period and in Patients 2 and 3 during thetwenty-first to thirtieth postburn day period. C5 con-

version by inulin in sera treated with MgEGTA was

markedly reduced in Patient 2 during the twenty-firstto thirtieth postburn day period, in Patient 3 until thethirty-fiFst to fortieth postburn day period, and in Pa-tient 1 for the duration of the study. C5 conversion insera treated with MgEGTA fell within the normal rangein Patient 4.

Levels and activities of classical pathway com-

ponents in the sera of the seven septic patients are

shown in Figure 5. In all of the patients, CH50 was

markedly reduced initially below the lowest value ob-tained in the nonseptic patients (67% of the normalmean value). Patient 6 was the only patient in whomclassical pathway components Clq, C4, C2, C3, andC5, as well as CH50, were reduced for the entire studyperiod of 21 days. This patient also had the most severe

bacterial infections with multiple micro-organismsisolated from all blood cultures which were obtainedafter the first day postburn. Patient 3 also had con-

sumption of Clq, C4, C2, C3, and CH50 during the 20day postburn period during which she was studied, ex-

cept during the sixth to tenth postburn day period; thefirst positive blood culture was not obtained on this pa-

tient until day 17. Patient 2 had multiple episodes ofsepticemia during 10-29 postburn days, associatedwith decrease in all of the classical pathway com-

ponents during this time. During the thirty-first tofortieth postburn day period, the classical complementcomponents except C3 were decreased in this patientand during the forty-first to fiftieth postburn day period,all of the components were restored to normal values.Patient 1, whose septic episodes occurred during days17-42, had decreased CH50, Clq, C4, C2, and C5, butnot C3, through the forty-first to fiftieth postburn dayperiod, which were normalized thereafter. In two of theother patients (#4 and 5), consumption of CH50, Clq,C4, C2, but not C3 or C5, occurred throughout thestudy except initially (days 13-15) in Patient 4, andduring the thirty-sixth to fortieth postburn day periodin Patient 5. During these times, all of the components,including C3 and C5, were decreased. Patient 4 hadseptic episodes on day 13 and days 31-34, and Patient5 became septic on day 37. The last patient (#7) had

40

30

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IN

520


C3 Conversion by Inulin (IC)

0~~~~~~~~~_ 4~~~~~~+2SC

_ ~~~~~~MEAh

50r'' " ' -2SI

I

) 1-5 6-10 11-20 21-30 31-40 41-50

C3 Conversion by Inulin in Untreated Burn Sera (H) C3 Conversion by Inulin in MgEGTA-Burn Sera (H)

/ ~ ~ ~~~~~~~~~~~~~~~~~~Hz Hemolytic\ / \ ~~~~~~~~~~~ICxlmmunochomical

+2SD +2SD.MEAN

D : -2SD MEAN

4 ~ ~

0 1-5 6-10 11-20 21-30 31-40 41-50 0 1-5 6-10 11-20 21-30 31-40 41-50

C; Conversion by Inulin in Untrea'ed Burn Sera (H) Con io Inulin in M -Burn Sera (H)

MEAN

0 1-5 6-10 11-20 21-30 31-40 41-50 0 1-56-10 11-20 21-30 31-40 41-50

DAYS POSTBURN

FIG. 4. Functional activity of the alternative complement pathway in the sera of four nonseptic burned patients during 50 days postburn. Con-version of C3 and C5 by inulin in untreated burn sera and in burn sera treated with 10 mM ethylene glycol tetraacetic acid and 10 mM MgCI2(MgEGTA-burn sera) was measured by hemolytic methods. Conversion of C3 by inulin and cobra venom factor (CoVF) in untreated burnsera was also measured by reduction in the B antigenic determinant of C3 by radial immunodiffusion. The shaded areas represent the variationin 20 individual normal sera (mean ± 2 S.D.). The Arabic numerals following the lines represent patient numbers. Refer to Table 2 for the clinicalcharacteristics of the patients. The points represent mean values of duplicate determinations.

CLASSICAL PATHWAY

0 >20 ar-30 1a 3-40 4-0 -40

ITIENT E

IN- 5-20 a-30 3-40 41-45

_ C3IC)O C3(H)EM C5IIC)

aZJ C5(H)IC ImmunochSmiCOIH HeoIytiC

I'.

PTIENT A

1DAY 1P1-OS-a

DAYS POSTBURN

FIG. 5. Immunochemical and functional activities of the classical complement pathway in the sera of seven septic burned patients during 50 dayspostburn. The levels of Clq, C4, and C2 were determined immunochemically. C3 and C5 were measured by immunochemical and hemolyticmethods. The Roman numerals represent the patient numbers. Refer to Table 1 for the clinical characteristics of the patients. The values ofduplicate determinations for each function are presented.

Vol. 189 * No. 4

200

150

100

-J

0zLA0

521

w-J41z4

r2-J4Ia:0zLL.0

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150 PAT"T

100

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intermittent septic episodes during 15-24 days post-burn; decrease in CH50, Clq, and C4, but not C2, C3, orC5, occurred during the entire study period of 30 dayspostburn.

Decrease in C3b INA was demonstrated in four ofthe seven patients (Fig. 6). C3b INA was decreasedduring the initial postburn period in three of the patients(#2, 3 and 4) and for the duration of the study in Pa-tient 6. C3b INA was also reduced in Patient 3 afterthe sixth to tenth postburn day period. Factor B wasdecreased concurrently with C3b INA except in Pa-tient 3. In this patient, factor B was normalized afterthe first five days postburn and remained normal there-after. Properdin was markedly reduced in all patientsfor the duration of the study.C3 conversion by inulin and CoVF, and C5 conver-

sion by inulin were reduced for the entire study periodin Patient 3, and until the thirty-sixth to fortieth post-burn day period in Patient 5 (Fig. 7). C3 conversionand C5 conversion were normal in Patient 6 until day21, probably because there were only small amounts ofC3 and C5 in this patient's sera to be converted by largeamounts of activating substances. In the other patients,

C3 conversion was often reduced when C5 conversionwas not and vice versa. In Patient 1, C3 conversion wasreduced for the entire study period, but C5 conversionwas normalized during the forty-first to fiftieth post-burn day period. In Patient 2, C3 conversion was re-duced during the entire study period except during theeleventh to twentieth postburn day period. C5 conver-sion in this patient was only reduced during the initialfive days postburn. In Patient 4, C5 conversion wasreduced for the entire study period of 50 days, but C3conversion was only reduced during 20 days postburn.In Patient 7, C3 conversion was reduced for the entirestudy period; however, C5 conversion was normal. Inall of the patients, C3 and CS conversion by inulin inthe burn sera was either not reduced or was onlyslightly reduced by treatment of the sera with Mg-EGTA.Reduction in complement levels and activities did

not decrease the ability of the patients' sera to pro-mote phagocytosis and intracellular killing of their in-fecting micro-organisms by normal human PMNs (Fig.8). Although S. aureus was the most common infectingmicro-organism, E. coli, Pseudomonas aeruginosa,

ALTERNAIIVE PATHWAY

200!ATIENT I

ISO

100

1501 ,1-SO2 21-30 31-40 41-0 5|1-3

PATIEN Z

* 13- IS1-20 .I-DON1

PATIENT K

PATIENT IL

4100

50

0 ~~5-10 Il-S SI~~-SO 5-40 41-3 S4 5SO 1- -0atL tt t 3

FACTOR B=1 C31b INA

. -40 41-SO

BPAIENT DC

FATIENT hI

I-S 6-10 I- is 14I40 i1 S -I0 l-SO SI-3O

DAYS POSTBURNFIG. 6. Immunochemical levels of factor B, properdin, and C3b inactivator (C3b INA) in the sera of seven septic burned patients during 50 dayspostburn. The Roman numerals represent the patient numbers. Refer to Table 1 for the clinical characteristics of the patients. The values ofduplicate determinations for each function are presented.

522

523SEPTIC BURNED PATIENT

ALTERNATIVE PATHWAY

PATIENT I PATIENT N150

0L 15-20 a-30 3-40 4-50 13- K-20 21-30 3-4 -

Ji PATIENT I PATIENT 9< 1s

z

w

00 0%z -5 6-10 11-20 21-30 31-40 41-5O 19-24 25-30 3-35

o PATIENT m150

01-5 6-60 11-15 16-20

m C3 Conversion by CoVFCl C3 Conversion by nlinin (IC)

C3 Conversion by Inulin (H)C3 Conversion by Inulin

in Mg EGTA-Bum Sero (H)C5 Conversion by Inulin (H)

_ C5 Conversion by Inulinin Mg EGTA Burn Sera (H)

IC= ImmunochemiclH =Hemolytic

PATIENT l PATIENT D9

11I-S6-10 Il-S 16-20 21 1-5 6-10 11-20 21-30

DAYS POSTBURNFIG. 7. Functional activity of the alternative complement pathway in the sera of seven septic burned patients during 50 days postburn. Conver-sion of C3 and C5 by inulin in untreated burn sera and in burn sera treated with 10 mM ethylene glycol tetraacetic acid and 10 mM MgCI2(MgEGTA-burn sera) was measured by hemolytic methods. Conversion of C3 by inulin and cobra venom factor (CoVF) was also measured byreduction in the B antigenic determinant of C3 by radial immunodiffusion. The Roman numerals represent the patient numbers. Refer to Table1 for the clinical characteristics of the patients. The values of duplicate determinations for each function are presented.

Klebsiella pneumoniae, Citrobacter freundii, andStreptococcus faecalis were isolated as well. In onlyone patient (#6), opsonic activity of the patient's sera

for her infecting strain ofK. pneumoniae was reduced;the reduction occurred only during the first five dayspostburn. It should be emphasized that none of the in-fecting micro-organisms was susceptible to direct lysisby normal or burn sera or to phagocytosis and intra-cellular killing by normal PMNs in the absence of thesera. The concentration of serum used in the opsonicassays was specific for each infecting micro-organism.

Concentrations of the patients' sera were based on theminimal concentration of pooled normal human serum

which was found to promote maximal intracellular kill-ing of each bacterial strain during the shortest incuba-tion period.5The types and amounts of blood products adminis-

tered to the septic patients are shown in Figure 9. Awide variation in the regimen for administration ofblood products was observed. All of the patients re-

ceived whole blood with Patients 1 and 2 receiving a

total of less than 1000 ml, Patients 3, 5, and 6 receivinga total of from 4000 to 6750ml, and Patients 4 and 7 re-

ceiving a total of 9000ml. Only four of seven septic pa-

tients received single donor plasma. Patient 3 received

the least amount (approximately 2000ml), and Patients4, 5, and 6 received from 7200 to 8500ml.

All of the nonseptic burned patients received singledonor plasma, and three of the four patients receivedwhole blood during the study period (Fig. 10). Patients1, 3, and 4 received from 250 to 2950ml of single donorplasma during the study; Patient 2 received the mostplasma, totaling approximately 8950ml. This patientalso received the most whole blood (12,485ml), with Pa-tients 3 and 4 receiving 795 and 4900ml respectively.Per unit of time, all of the nonseptic patients with theexception of Patient 2 received less whole blood andplasma than the four septic patients who received bothblood products.

Discussion

The data presented in this investigation support our

previous preliminary observations that consumptionof the classical complement pathway was associatedwith and was caused by septicemia in thermally in-jured patients.2 In this investigation, all of the seven

study patients had decreased classical pathway activityduring their septic episodes, and there were no excep-

tions. All of the patients who did not survive septicemia

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PATIENT I

15-20 21-30 31-40 41-50 51-60

PATIENT II

PATIENT m

PATIENT X

100- N.M.U

50

13-15 16-20 21-30 31-40 41-50

PATIENT v

100 _ N.M.-

501-

19-24 25-30 31-35 36-40

S.aureusO E.coliCM P.aeruainosa! Koneumoniae89 S.foecalis

C.freundii

N.M.=Normal Mean

PATIENT

PATIENT M

1-5 6-10 11-15 16-20 1-5 6-10 11-15 16-20

FIG. 8. Serum opsonizing activity for the micro-organisms causing septicemia in the seven burned patients during 50 days postburn. None of themicro-organisms isolated from the patients and used in the opsonic assays was susceptible to killing by either leukocytes or serum alone. Serumconcentrations in the assays varied for each micro-organism and were based on the minimal concentration ofpooled normal human serum whichpromoted maximal intracellular killing of the micro-organism during the shortest incubation period. The Roman numerals represent the patientnumbers. Refer to Table 1 for the clinical characteristics of the patients. The values of duplicate determinations for each function are presented.N.M. represents the normal mean value.

and who died of septic shock had consumption of theclassical complement components (Cl-C5) duringtheir septic episodes. Patients who survived septicemiahad multiple patterns of classical complement pathwayconsumption during their septic episodes as follows: a)Consumption of Cl-C5, b) consumption of only Cland C4, c) consumption of only C1, C4, and C2, and d)consumption of C1, C4, C2, and C5, but not C3. Inthese patients, classical pathway activity was restoredto normal within 20 days following the last positiveblood culture. This observation is preliminary, how-ever, since only two of the four surviving patients werestudied long enough following septicemia to make thisevaluation.The alternative complement pathway was consumed

in only one of the seven septic patients. C3b INA andfactor B were markedly decreased for the entire studyperiod in this patient. The results suggested that al-ternative pathway consumption occurring during sep-ticemia in this patient resulted from the generation of

C3b via consumption of the classical pathway. C3btogether with factors B and D, and properdin wouldlead to further depletion of C3 and C5 via the enzymes,C3bBb and C3bBbP. This observation provides an

explanation for the results of our earlier study, show-ing that both complement pathways were consumed inone septic burned patient, and only the classical path-way was consumed in the other septic patient.1The classical pathway appeared to be activated pref-

erentially in all of the other burned patients during sep-tic episodes. Although C3 and/or C5 conversionwere often reduced during septic episodes, factor B was

decreased in only three of the patients during the firstfive days postadmission. C3b INA was also decreasedin these patients, and in two of them, occurred onlywhen C3 was reduced concurrently. The decrease inC3b INA appeared to result from increased utiliza-tion in cleavage of C3b to its inactive forms. Since it iswell known that factor B is consumed during alterna-tive pathway activation, the results suggested that the

100

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524 Ann. Surg. 9 April 1979


PATIENT J

PATIENT I

5000-

4000-

3000-

2000 -

1000

a a15-20 21-30 31-40 41-50 51-60

PATIENT I[

1-5 6-10 1-20 11-30 31-4 41-50

I] I13-15 16-20 21-30 31-40 41-50

PATIENT v

PATIENT m20C

10c

1-5 6-10 11-IS 6-20

* PATIENT ME

101-5 I6-10111 6-20 24

2000 _

000 _

I-S 6-10 11-20 2-30 31-40

FIG. 9. Temporal sequence of the administration of blood products to the seven septic burned patients. The Roman numerals represent the pa-

tient numbers. Refer to Table 1 for the clinical characteristics of the patients.

alternative pathway was reduced in the burned patientsdue to blockage of this pathway rather than to con-

sumption of alternative pathway components.Reduction in classical pathway activity was also dem-

onstrated in all of the septic burned patients duringthe initial postburn period and during the time prior towhen the first positive blood culture was obtained.Total hemolytic complement (CH50) was always mark-edly reduced below the normal mean value for the non-

septic patients, and early complement componentswere reduced as well. The reduction in classical path-way activity was not found to be the result of systemicinfection, since blood cultures were consistently nega-

tive in the patients during this time. Reduction inclassical pathway activity was not demonstrated in thenonseptic burned patients, suggesting that this humoralabnormality is predictive of septic episodes. This is anextremely exciting observation and should lead to theearly identification of high risk patients and possiblealterations in therapeutic approaches.The lack of demonstration of classical complement

pathway consumption during the study period in thenonseptic burned patients could not be explained on the

basis of the administration of blood products, sincethese patients as a group received less whole blood andplasma than the septic burned patients. The administra-tion of blood products also did not influence the out-come of septicemia, since the patient with the most pro-

longed septicemia received the largest amounts ofblood products per unit of time. In addition, the twoother patients who died of septic shock receivedamounts of whole blood and plasma per unit of timeroughly equal to the nonseptic patient who received themost blood products.More abnormalities of C3 and C5 conversion by

CoVF and/or inulin were observed in the septic burnedpatients than in the nonseptic burned patients. How-ever, there was quite a discrepancy in the average burnratio (per cent total injury/per cent third-degree in-jury) between the two groups (55/43 for the septic pa-

tients and 49/19 for the nonseptic patients). The in-creased number of abnormalities in the septic popula-tion is probably explained by this finding, since reduc-tion in C3 conversion has been shown to be a functionof increasing burn size.' 2 C3 and C5 conversion by theactivating substances was shown to be a function of the

Vol. 189 * No. 4 525

o-~

200

100

0

w

cr

z

n

D 20C0

0

0rlo

0

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O PLASMA* WHOLE BLOOD* PACKED CELLS

PATIENT 3ZM


Patient I2000-

1000 -

1-5 6-10 11-20

Patient 2

0

21-30 31-40 41-50

4000 -

3000 -

2000

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Patient 3

n 1I II13-15 16-20 21-30 31-40 41-50

Patient 4

* Whole BloodSingle Donor Plasma

El Packed Cells

I Il1-5 6-10 11-20 21-30 31-40 41-50

DAYS POSTBURNFIG. 10. Temporal sequence of the administration of blood products to the four nonseptic burned patients. The Arabic numerals represent thepatient numbers. Refer to Table 2 for the clinical characteristics of the patients.

alternative complement pathway. Values for con-

centrations and conversion of C3 and C5 were con-

sistently higher when measured by hemolytic assays,in comparison to the results obtained from the im-munochemical determinations. In addition, a markedvariability in the results of the hemolytic assays was

demonstrated. However, the most important observa-tion to be derived from the C3 and C5 conversion datais that there was absolutely no correlation betweenthese abnormalities and the occurrence, duration, or

outcome of septicemia.Consumption of components of the alternative and/

or classical complement pathway did not decrease theopsonic capacity of the patients' sera for their own in-fecting micro-organisms. The investigators offer twopossible explanations for this observation as follows: 1)immune antibodies may be produced during the infec-tion which either alone or in combination with minimallevels of classical complement components effectivelyopsonize the infecting micro-organisms; or 2) naturallyoccurring antibodies to the infecting micro-organismsmay be present in the burn sera prior to the infectionwhich together with minimal levels of alternative and/orclassical complement pathway components effectively

opsonize the micro-organisms. Data have been pre-sented which show that under normal conditions the ma-jority of gram negative aerobic bacilli isolated fromburned patients require immunoglobulin and utilize thealternative and/or classical complement pathways dur-ing the opsonic process.13 Therefore, either antibodiesmust be present in the burn sera prior to infection or beproduced during colonization with the micro-or-ganisms, if effective opsonization in the presence ofminimal levels of complement components is to occur.

It could be speculated that the antibodies are presentin the burn sera prior to infection as a result of the ad-ministration of blood products. In this regard, Saba etal.1I have recently reported that administration offreshplasma cryoprecipitate to septic trauma patients cor-

rected a deficiency in an a2 glycoprotein formerly desig-nated opsonic protein. It should be emphasized that thebiological significance of this protein in opsonization ofmicro-organisms is unknown. Our future studies will bedirected toward determining the levels of pre-existingantibodies and antibodies produced during the course

of the infection to the infecting micro-organisms inseptic burned patients. The septic burned patient repre-sents a new model for studying the in vivo role of com-

2000 -

E 1000 -

Ct)Lj 0

4000 -

K 3000 -

2000-

a. 1000-

0

526 Ann. Surg. * April 1979

I II


plement and immunoglobulins in opsonization of op-portunist micro-organisms.

AcknowledgmentsThe authors express their appreciation to Dr. Clark West,

Childrens Hospital Research Foundation, Cincinnati, Ohio forreference sera and antisera to human complement components. Theexcellent technical assistance of Ms. Barbara Kitko, Ms. RebeccaMiller, and Ms. Mary Lou Iserson is gratefully acknowledged.

References1. Bjornson, A. B., Altemeier, W. A. and Bjornson, H. S.:

Changes in Humoral Components of Host Defense FollowingBurn Trauma. Ann. Surg., 186:88, 1977.

2. Bjornson, A. B., Altemeier, W. A., Bjornson, H. S., et al.: HostDefense against Opportunist Microorganisms FollowingTrauma. I. Studies to Determine the Association BetweenChanges in Humoral Components of Host Defense and Sep-ticemia in Burned Patients. Ann. Surg., 188:93, 1978.

3. Fine, D. P., Marney, S. R., Jr., Colley, D. G., et al.: C3 ShuntActivation in Human Serum Chelated with EGTA. J.Immunol., 109:807, 1972.

4. Gotze, 0. and Muller-Eberhard, H. G.: The C3-Activator Sys-tem: An Alternate Pathway of Complement Activation. J.Exp. Med., 134:90S, 1971.

5. Hirsch, J. G. and Strauss, B.: Studies on Heat-Labile Opsoninin Rabbit Serum. J. Immunol., 92:145, 1964.

5276. Leist, P. A. and Bjornson, A. B.: Heterogeneity of Opsonic

Requirements for Gram-Negative Aerobic Bacilli, Abstractsof the 78th Annual Meeting of the American Society forMicrobiology, p. 188, 1978.

7. Mancini, G., Carbonara, A. 0. and Heremans, J. F.: Immuno-chemical Quantitation of Antigens by Single Radial Immuno-diffusion. Immunochemistry, 2:235, 1965.

8. Mayer, M. M.: Complement and complement fixation. InExperimental Immunochemistry, second edition. Springfield,Charles C Thomas. 1971. p. 162.

9. Nelson, R. A., Jensen, J., Gigli, I. and Tamura, N.: Methodsfor the Separation, Purification, and Measurement of NineComponents of Hemolytic Complement in Guinea Pig Serum.Immunochemistry, 3:111, 1966.

10. Pensky, J., Hinz, G. F., Jr., Todd, E. W. and Pillemer, L.:Properties of Highly Purified Properdin. J. Immunol., 100:142, 1968.

11. Ruley, E. J., Forristal, J., Davis, N. C., et al.: Hypocomple-mentemia of Membranoproliferative Nephritis. Dependenceof the Nephritic Factor Reaction on Factor B. J. Clin. Invest.,52:8%, 1973.

12. Saba, T. M., Blumenstock, F. A., Scovill, W. A. and Bernard,H.: Cryoprecipitate Reversal of Opsonic a2-Surface BindingGlycoprotein Deficiency in Septic Surgical and Trauma Pa-tients. Science, 201:622, 1978.

13. Todd, E. W., Pillemer, L. and Lepow, I. H.: The ProperdinSystem and Immunity. IX. Studies on the Purification ofHuman Properdin. J. Immunol., 83:418, 1959.

14. West, C. D., Davis, N. C., Forristal, J., et al.: Antigenic Deter-minants of 13lC-globulin. J. Immunol., 96:650, 1966.

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the septic burned patient

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