~

WILLlAM BROWDER, M.D., DAVID WILLlAMS, PH.D., HENRY PRETUS, PH.D., GIORGIO OLlVERO, M.D.,FRANCESCO ENRICHENS, M.D., PATRIZIO MAO, M.D., and ALLESSANDRO FRANCHELLO, M.D.

Host immunosuppression after trauma contributes to septicmorbidity. The macrophage is a key element in the host immuneresponse. This study evaluated glucan, a macrophage stimulant,in a prospective, randomized, double-blind study of 38 traumapatients undergoing surgery. Glucan (21 patients), 50 mg/m2,or placebo (17 patients) ,,'as given intravenously daily for 7 days.Delayed hypersensiti\ity skin testing was performed on days 1and 7 after trauma. Serum interleukin-1 (IL-I) and tumor ne-crasis factor (TNF) ,,'ere assayed after trauma. While the totalmortality rate ,,'as significantly less in the glucan group (0%versus 29%) (p < 0.05), the mortality rafe from sepsis ,,'as notstatistically different (0% versus 17.6%). Glucan therapy signif-icantly decreased septic morbidity (9.5% versus 49%; p < 0.05).Serum IL-I had a greater increase in glucan patients on dar 3after trauma (143.4 :!: 19.3% versus 78.6 :!: 11.7%; p < 0.05),but there ,,'as no difference thereafter. Serum TNF did not varybet,,'een groups. Early increase in IL-I correlated with subse-quent skin test com'ersion to positive. Neither serum IL-I norTNF "'as a reliable indicator of roture sepsis. Further clinicaltrials are indicated to e\'aluate biologic response modifiers thatactivate macrophages in the trauma patient.

From the Departments of Surgery and Physiology, TulaneUniversity School of Medicine, New Orleans, Louisiana; and

Istituto Di Chirurgia D'Urgenza, Universityof Torino, Torino, Italy

tigen processing and presentation by the macrophage,which is essential for proper lymphocyte response, is sup-pressed after hemorrhage7 and tissue trauma.8 Macro-phage secretory products such as interleukin-l (IL-l) andinterferon, which are important mediators of immunedefense, are decreased after trauma.9 In response totrauma, so-called inhibitory macrophages produce pros-taglandin E2 (PGE2), which is capable of suppressing avariety ofimmune functions.1o In addition serum opsoninlevels and reticuloendothelial clearance of bacteria aredepressed in the trauma patient.ll

Given the central role ofthe macrophage in mediatingthe overall immune response, a reasonable approach tothe amelioration of immune suppression in the traumapatient might include biologic response modifiers (BRM)that enhance macrophage function. Our laboratory hasextensively studied glucan, a potent macrophage stimulantthat has been shown to be beneficial in animal models ofsepsis,12,13 trauma,14 and wound healing.15 The presentstudy was designed to assess the value of glucan in a pro-spective trial of trauma patients. In addition the mono-kines IL-I and tumor necrosis factor (TNF), which maybe important in maintaining host-immune responsive-ness,16-18 were measured in the early post-trauma period,and correlated with delayed hypersensitivity skin testingand the subsequent development of sepsis.

T HE PAST DECADE has witnessed majar advancesin the care ofthe trauma patient. Improved fieldresuscitation and rapid transport to levell trauma

centers have resulted in improved early survival aftertraumatic injury. However subsequent morbidity andmortality related to sepsis are still majar problems in thecomplete recovery ofthe trauma patient. A primary factorin the development ofsepsis is depression ofhost-immuneresponse after severe injury. Previous studies have doc-umented that both cellularl-3 and humoral4-6 immunityare diminished in the patient with severe injury. Alteredmacrophage function is reported to be a key element inthe increased susceptibility to sepsis after trauma!.8 An-

Methods

~

Presented at the 10lst Annual Meeting ofthe Southern Surgical As-sociation, Hot Springs, Virginia, December 3-6, 1989.

Address reprint requests to Dr. William Browder, Tulane UniversityDepartment ofSurgery, 1430 Tulane Avenue, New Orleans, LA 70112.

Accepted for publication January 19, 1990.

Palienl Populalion

The Trauma Institute in Torino, Italy is the equivalentof a level l trauma center and is the only such facility

605

606 BROWDER AND OTHERS Ann. Sur¡.. May 1990

Blood Analysis

Blood was'collected in siliconized giass tubes on days1, 3, 5, 7, and 9 after trauma for determination of serumIL-I levels. Similarly blood was collected on days 2, 4, 6,8, and 10 for tumor necrosis factor assay. Leukocytecounts were determined daily for the first week after op-erative intervention. Serum for IL-I .B and TNF-a deter-minations was stored frozen in plastic vials until assayed.

Radioimmunoassay o/ IL-l .8 and TNF-a

Serum levels of IL-1 fJ or TNF-a were determined byradioimmunoassay using commercial1y available kits (1251-IL- 1, RIA kit, Advanced Magnetics, Cambridge, MA and1251-TNF-TEST, Genzyme Corp., Boston, MA, respec-tively). For either assay, a IOO-JLL aliquot ofunextractedserum was assayed directly, and al1 samples were ron induplicate. Cross reactivity for the antibody included ineach kit was negligible for other cytokines.

Statistical Analysis

Statistical comparisons were performed by one-wayanalysis of variance where appropriate. Differences be-tween groups were determined using a Bonferroni mul-tiple comparison test. Additional comparisons were con-ducted using chi-square with Yates correction and theStudent's t test.

Statistical significance was assumed at p < 0.05. Sta-tistical studies were performed using STA T A StatisticalSoftware Package (Computing Resource Center, Los An-geles, CA) ron on an IBM-compatible personal computer.

Results

serying approximately 1.2 million people. The study wasdesigned to evaluate patients admitted to the Trauma In-stitute between the ages 18 and 65 years who underwentexploratory laparotomy or thoracotomy for trauma. Ex-cluded patients included those with cirrhosis, renal failure,severe head injury, or reproductive potential. Eligible pa-tients were entered joto a prospective, randomized, dou-ble-blind study to receive either glucan (50mgtm1 or sa-line placebo intravenously daily for 7 days after operation.This dose of glucan has been demonstrated to increaseinterleukin-l and interleukin-2 production in humans.19All colon injuries were treated with colostomy and delayedprimary wound closure. AII patients received prophylacticantibiotics, usually a cephalosporin, for 5 to 7 days. Sub-sequent antibiotic therapy was contingent on signs ofclinical infection, as well as appropriate culture and sen-sitivity results. Patients underwent intraoperative auto-transfusion as indicated. Parenteral nutrition was begunimmediately after operation in all patients and continueduntil bowel function returned. Postoperative ventilatorsupport was used in all patients. Weaning from the ven-tilator and subsequent extubation was scheduled on anindividual patient basis. An index of injury severity wasestablished by calculating the Injury Severity Score(ISSfo,21 Clnd the Abdominal Trauma Index,22 as previ-ously described.

Skin tests were applied on days 1 and 7 after traumausing the Multi- Test device,23 which uses seven antigens(tetanus, diphtheria, streptokinase/dornase, tuburculin,candidin, tricophytin, and proteus) and one control.Anergy was judged to be less iban two reactions of 2 mminduration.24

A clinical infection was documented when the patient'stemperature rase above 39 C daily and there was evidenceof majar soft-tissue involvement with positive bacterialcultures (wound, lung, or abdomen).

This clinical protocol was approved by both the Com-mittee on Use of Human Subjects at Tulane Universityand the Institutional Review Board ofthe Trauma Insti-tute, University of Torino, Torino, Italy. Informed con-sent was obtained from all patients or their immediatenext-of-kin.

Glucan Preparation

Soluble glucan was prepared in the Departments ofSurgery and Physiology laboratories, Tulane UniversitySchool of Medicine. Particulate glucan was prepared bya modification ora previously described procedure!5 Sol-uble glucan was prepared according to the process de-sct:ibed in United States Patent 4,739,046. The glucanwas shown to be endotoxin free as determined bythe Limulus Iysate procedure (Sigma Chemical Co., StoLouis, MO).

From 1986 to 1988, a total of 38 patients were enteredin the study (21 glucan, 17 placebo). There were 19 maleand 2 female patients in the glucan group and 12 maleand 5 female patients in the placebo group. Blunt traumadue to automobile accident was the most common modeof injury, accounting for 12 of the 21 injuries in the glucangroup and 15 of the 17 injuries in the placebo group.Penetrating injuries due to gunshot or stab wounds ac-counted for the majority ofthe remaining patients. Whilethe glucan-treated patients had injuries to the lung andcolon, the placebo group had more liver and duodenalinjuries. However there was no statistically significant dif-ference in the distribution of organ injuries between thetwo groups (Table 1).

A comparison of injury severity in the two groups ofpatients is listed in Table 2. Injury severity and A TI scoreswere similar. In addition there was no significant differencebetween the two groups in the amount of blood transfu-sion perioperatively or in the number of patients pre-

MACROPHAGE ACTIVA TION IN TRAUMA 607VoI.211.No.S

TABLE Breakdoll'n alargan Injury in 38 Trauma Pafienfs Admilfedlo lhe Clinical PrOloco/"

T ABLE 3. Description o/ Septic Morbidity in Trauma PatientsReceiving Placebo or Glucan Treatment

Placebo Glucan ComplicationOrgan Injured Placebo Patients Glucan Patients p Value

84233

5765O

Abdominal abscessPneumoniaWound infectionGeneralized sepsisTotal

o2OO2

42

nos.nos.n.s.n.s.

<0.0518I

I o

.There was no significant variation between placebo and glucan pa-tients with regard to organ injury. Neither the ISS nor the A TI correlated with develop-

ment of sepsis. However those patients developing sepsisdid receive a greater amount of blood transfusion whencompared to those who did not have septic complications(1333.3 :t 795.3 cc versus 934.4 :t 398.4 cc; P < 0.05).

senting to the emergency department with systolic bloodpressure less than 90 mm Hg.

Serum IL-I fJ and Serum TNF a AssaysDelayed H)persensitivity

In this protocol, patients who were anergic on dar 7,regardless of treatment group, had a greater incidence ofsubsequent sepsis than reactive patients (53.3% versus8.7%; p < 0.0 1). A total of 14 placebo patients and 15gIucan patients showed negative skin testing on dar 1 aftersurgery. However the gIucan-treated patients had a sta-tistically significant increase in conversion to positive skintesting on dar 7 when compared to the placebo group(73% versus 21%; p < 0.02).

The percentage increase in serum IL-llevels for glucanand placebo patients is shown in Figure l. The glucanpatients had a significant increase in IL-l on day three(143.4% ::t 19.3% versus 78.6% ::t 11.7%; p < 0.05). Byday five and thereafter, there was no difference in the IL-1 levels between the two groups. The early increase in IL-1 in glucan patients correlated with subsequent skin testconversion on day 7 (Fig. 2). Glucan patients who con-verted to positive skin testing had a significantly greaterincrease in IL-l levels (166.8% ::t 12.4% versus 119.8%::t 4.2%; p < 0.02).

There was no difference in the serum TNF levels ofplacebo- or glucan-treated 'patients (Fig. 3). In additionthere was no correlation between the serum IL-l or TNFlevels alone and the subsequent development of sepsis,regardless ofplacebo or glucan therapy (Fig. 4).

.p ( 0.05 n placebo

Morbidity and Mortality

Total mortality was significantly less in the glucan groupwhen compared to placebo (0% versus 29%; p < 0.05).However two ofthe placebo deaths were cardiac arrhyth-mias secondary to a pulmonary embolus and myocardialinfarction. The three placebo deaths due to sepsis (17.6%)were not significantly greater iban the zero mortality inthe glucan group. The three placebo deaths from sepsiswere secondary to abdominal abscess (2) and pneumo-nia (1).

Morbidity from sepsis was significantly greater in theplacebo group (49% versus 9.5%; p < 0.05). Specific mor-bidity is shown in Table 3. The four abdominal abscesseswere secondary to duodenal (2), hepatic (1), or bowel in-juries (1), and allweré1reatéd with reoperation, with twosubsequent deaths.

/."""" 1

I~-':;<'

TABlE 2. lnjury Severity in Glucan and Placebo Patients

Placebo Patients Glucan PatientsFactor

32.926 (14.4 (

28.6%5.0

35.7 years23 (8-34)15.8 (8-26)

AgeISS.ATItAdmission hypotension

(BP < 90 mm/Hg)Amount ofblood transfusion

175-

150-

>, 125~O 100O 75-

~50-

o-o Placebo25. e-e Glucan

O! O 1 2 3 4 5 6 7 8

Iadmiasion TIME (days)

F1G. l. Elevated serum IL-I (3 levels in glucan-treated trauma patients.Serum was obtained from placebo and glucan patients on days 1, 3, 5,and 7 after trauma. IL-I (3 was quantitated by radioimmunoassay. Asignificant increase in IL-I (3 was observed in glucan-treated trauma pa-tients on day 3.

41% (7/17)5.3 units

.Injury Severity Score.t Abdominal Trauma Index,

I

years16-41)0-35)

(6/21)units

~~

608 BROWDER AND OTHERS Ann. SUI¡. .May 1990

200:

175J

'::;' 150.1>.G 125-A

"O 100:.

~ 75JI

50.

25~

0-

150."", I -...J >-

Ge Q 100-

~ -o'Qj ~cn '-' 50.

Serum IL-l P Serum TNFa

AG. 4. Lack of correlation between serum IL-! .B or TNFa and deve!-opment of septic morbidity in trauma patients. IL-! .B and TNFa werequantitated by radioimmunoassay.

Anergic Conversion

Skin Test ConversionAG. 2. Positive correlation between elevated serum IL-1 {J levels andskin test conversion in trauma patients treated with glucan. The increasein IL-1 {J was significantly greater (p < 0.05) in glucan-treated traumapatients who con verted as opposed to those who remained anergic. Ski ntesting was undertaken on days I and 7 ~ith a Multi- Test device usingseven antigens.

Leukocyte Count

Peripheralleukocyte counts for the two groups duringthe first week after operation are presented in Figure 5.While the mean white blood cell counts in the glucan.treated patients were higher on each ofthe days, the widevariation precluded statistical significance in ibis study.

relative increase in the subset of suppressor lymphocy1es.1Polymorphonuclear leukocy1e (PMN) function is also de-pressed, as denoted by decreased chemotaxis, phagocy-tosis, and intracellular killing in PMNs isolated from thetrauma patient!6,27

Humoral immunity is also altered, as retlected by re-duced serum immunoglobulins after severe trauma.4,28Similarly altered nonspecific immunity in the form of fi-bronectin-mediated clearance of blood-born particulatematter by the reticuloendothelial system has been impli-cated in the pathogenesis of sepsis after severe trauma.29Specifically depleted fibronectin and subsequent di mi n-ished blood clearance has led to multiple-organ failureand sepsis in the severely injured patient.29,30

Recent years have seen the macrophage emerge as akey cell in immune dynamics after trauma. Macrophageproduction of cy1okines that participate in virtually allaspects of cellular and humoral immunity make it an im-portant mediator ofhost-immune response. Therefore anyalteration in macrophage function can significantly affect

Discussion

Severe trauma has a major impact on homeostasis. Inaddition to derangements in endocrine and metabolicfunctions, there is severe depression of host-immune re-sponse. Severa! studies have documented such suppressionoccurring in both humoral and cell-mediated immuneresponses.I,26,27 Lymphocyte activity, as measured by mi-togen responsiveness, is decreased.1 In addition there is a

o 1 2 3 4 5 6

Time (days alter admission)

AG. 5. Daily peripheralleukoc}1e counts in trauma patients after placeboor glucan treatment. There was no significant difference between treat-ment groups.

~

FIG. 3. Serum TNFa levels in trauma patients after placero or glucantreatment. Serum was collected on days 2, 4, 6, and 8 after trauma.TNFa was quantitated by radioimmunoassay. There was no significantdifference between treatment groups.

MACROPHAGE ACTIVATION IN TRAUMA 609

creased, contributing to improved immune responsive-ness.40

In the present study, glucan-treated patients had a sig-nificant increase in IL-l levels on day 3 "after trauma.However by day 5 and thereafter, there was no differencein IL-llevels between t:he two groups. The immune statusof the trauma patient in this early period may be criticalfor subsequent development of sepsis. Faist et al.lo re-ported suppressed in vitro mononuclear cell function ondays 5 to 7 after trauma. This depression correlated withdevelopment of subsequent infectious complications.JOSimilarly, Rodrick et al.9 studied mononuclear cells fromtrauma patients after operation. There was a significantdecrease in IL-l production from days 1 through 5 aftertrauma, with a subsequent return to the normal range.9Stephan et al.s used a murine model to study membraneIL-l activity of peritoneal macrophages after laparotomy.They noted a marked depression of IL-l activity on days1 and 3 after laparotomy, with activity returning to normalby day 5 after trauma.s It is apparent that macrophagedepression early in the period following severe traumamay be a majar factor in subsequent development of sep-siso It is encouraging that in these studies IL-l production

.was significantly greater after glucan therapy i~ the earlypost-trauma periodo Why glucan therapy was unable toenhance IL-llevels beyond day 3 after trauma is unclear.However this may relate to the dose of glucan used inthese patients. Further studies are necessary to better de-lineate dose-response curves for glucan therapy.

Those glucan patients whocconverted from a negativeskin test on day 1 to a positÍve skin test on day 7 aftertrauma had a significantly increased IL-l level whencompared to the glucan patients who did not convert.The macrophage is a key cell in delayed hypersensitivityand the positive correlation between IL-llevels and skin-test reactivity further supports the concept of macrophage~ctivation in these patients. Skin test results also correlatedwith development of subsequent septic complications, inagreement with many previous studies.42-44 However inthe present study measurement of serum IL-l alone did

predict subsequent septic complications.This prospective randomized study resulted in very

comparable study groups. While there was some vari-ability inorgan injuries, overall trauma severity as assessedby ISS and A TI was very similar. In addition there wasno significant difference between groups in the incidenceofhypotension or the amount ofblood transfused. Whileoverall m ortal it y was significantly decreased in the glucan-treated patients, two ofthe five deaths in the placebo groupwere cardiac in nature and unrelated to sepsis. The threedeaths from sepsis in the placebo group (versus Done inthe glucan-treated patients), while not statistically signif-icant, certainly suggest that enhanced macrophage func-tion imparts some protection to the trauma patient. The

Vol.211.No.5

iromunologic responsiveness. An intriguing concept de-scribes two general classes of macrophages: facilitory andinhibitory?6.31.32 Facilitory macrophages are noted for thepresence of la antigen and the production of stimulatorymonokines that interact with T -helper lymphocytes. In-hibitory macrophages, on the other hand, are recognizedlI>y their lack orla antigen and production ofprostaglandinE2 (PGE2), which interacts with T -suppressor lympho-~ytes.1O.26.31.32 In particular the production of PGE2 byinhibitory macrophages may represent an important im-munosuppressive mechanism because these cells are in-

~reased after traumatic injury.33,34 Among PGE2s manyoimmunosuppressive effects, augmentation ofT -suppressortell proliferation, and inhibition of T -helper and B-cellproliferation have been well documented.33 In additionPGE2 inhibits production of certain cytokines such as IL-1 and IL-2, which are important for proper T -lymphocyteresponse.33 It is apparent that PGE2 release by inhibitorytnacrophages may playa major role in post-traumatic im-~unosuppression. Several animal studies have docu-rnented the beneficial effects of ibuprofen, a cycloxygenaseinhibitor, in models of traumatic and postburn sepsis.lnhibition of prostaglandin synthesis by ibuprofen in thesernodels resulted in increased survival.3s,36

A possible therapeutic approach to post-traumatic im-rnunosuppression might involve the concept of selectiveincrease and enhancement of facilitory macrophages.Previous studies have demonstrated that glucan, a beta-l, 3-linked glucopyranose polymer, isolated from the innercell wall of Saccharomyces cerevisiae, is a potent mac-rophage stimulant and is beneficial in the therapy of ex-perimental bacterial, viral, and fungal diseases.12,13,37,38Use of glucan in a murine model ofhind-limb crush injurydecreased macrophage PGE2 release while stimulatingbone marrow proliferation.14 This reversal ofmacrophagefunction toward the facilitory state might be of majarbenefit to the trauma patient. In addition glucan is watersoluble and nontoxic and is thus suitable for parenteraladministration in humans.39

As noted previously macrophage production of IL-I isan indicator ofthe activated state ofthe macrophage.13,ISSeveral studies have demonstrated that macrophage ac-t.ivation by glucan results in a marked increase in IL-Iprüduction.13,IS Intravenous glucan administration inhumans has likewise resulted in pronounced increases inIL-l and IL-2.19 In addition to reflecting the functional~tatus of the macrophage, IL-l may exert protective effectsin the trauma patient. IL-l activates lymphoc)l1eS and'promotes lymphocyte release of IL-2, with resulting en-hanced lymphocyte number and function.4O IL-l also en-

hances mobilization of polymorphonuclear leukocytesfrom bone marrow and enhances their chemotactic abil-ity.41 IL-l triggers the acute-phase response in which he-patic synthesis of certain proteins is dramatically in-

---,,"!~

610 BROWOER ANO OTHERS Ann. Surg.. May 1990

overall incidence of sepsis was significantly greater in theplacebo group. More significant were the four abdominalabscesses noted in the placero group versus Done in thegIucan-treated patients. Two ofthe three septic deaths inthe placero group resulted from multiple end organ failureafter persistent abdominal sepsis unresponsive to surgicaldrainage and antibiotic therapy. Similar results have beenreported by Meakins et al.,45 who performed a double-blind study in surgicaI patients using levamisole to preventinfection in those high-risk patients who were anergic orrelatively anergic to delayed hypersensitivity skin testing.While overall mortality was not different in the twogroups, levamisole recipients did haye significantly fewerseptic complications. This clinical success was accom-panied by improved neutrophil chemotaxis and skin. test

reactivity.45Previous studies have demonstrated that systemic ad-

mil1istration of gIucan mar impart protective effects inthe peritoneaI cavity.12,13,46 In animal models of Esche-richia coli peritonitis, gIucan therapy increased the num-ber and function of macrophages in the peritoneal cav-ity.12,13 In addition PMN number and function were in-creased peripheraIly as well as in the peritoneal cavity.12.46Thus gIucan therapy in the immediate post-traumatic pe-riod mar mobilize monocyte and PMN celllines to en-hance antimicrobiaI defenses in the peritoneal cavity.

Recent studies have focused on TNF and its role as amediator in a variety of pathologic states.47.48 There isgood evidence that TNF produces the metabolic, cardio-vascular, and inflammatory changes seen with endotoxin.Tumor necrosis factor mar represent an essentiaI mediatorin the complex pathophysiology of endotoxic shock.47.48However there is evidence that TNF mar impart someprotection in septic patients. Tumor necrosis factor hasbeen documented to increase both polymorphonuclearleukocyte and monocyte cytotoxicityl7,49 and mar influ-ence release of other cytokines, especially IL-l and gran-ulocyte macrophage-colony stimulating factor (GM-CSF).16,50 Livingston et al.16 have studied TNF in com-bination with antibiotics in a murine model of infectionfollowing hemorrhagic shock. While TNF alone did notreduce infection, its addition to the antibiotic regimensignificantly improved results when compared to the an-tibiotic alone.16 It is obvious that the ultimate effect ofTNF on the host is dependent on both the dose of TNFand the interaction ofTNF with other mediators presentin the biologic milieu. In the current study, gIucan therapydid not result in an increased level of serum TNF. Fur-thermore serum TNF did not correlate with subsequentdevelopment of sepsis. Further studies are needed to betterdelineate the role of TNF in the development of post-traumatic sepsis.

-The amount of perioperative blood transfusion in thetwo treatment groups was the same in this study. However

those patients who subsequently developed sepsis receivedsignificantly more transfused blood than did those patientswho had no séptic morbidity. This is not surprising be-cause there is increasing evidence that blood transfusionis immunosuppressive.51.52 Many studies have docu-mented that the macrophage may be a key factor inthis post-transfusion immunosuppression.52-54 Decreasedlymphocyteresponsiveness in post-transfusion patientshas been related to impaired macrophage lymphocyte in-teractions, possibly mediated by prostaglandin E2 releasedfrom the inhibitory macrophage.52.53 Macrophage che-motaxis and phagocytosis are also impaired after bloodtransfusions.53 Several studies have linked post-transfusionimmunosuppression to increased susceptibility to infec-tion.54.55 Wound innoculation with Pseudomonas aeru-ginosa in a rat bum model resulted in higher mortalityTales when rats were resuscitated with allogeneic blood asopposed to saline or syngeneic blood transfusion.54 It hasalso been demonstrated that survival in a rat E. coli peri-tonitis model was significantly reduced after allogeneicblood transfusion.55 It is apparent that biologic responsemodifiers that enhance macrophage function may playaTole in ameliorating or preventing immunosuppressionafter blood transfusion. Findings in this study supportthat concepto

A potential protective mechanism with glucan stimu-lation is macrophage release of GM-CSF. This phenom-enon has striking effects on boDe marrow proliferation.Patchen et al.56,5? have demonstrated that glucan therapywill significantly accelerate hematopoetic recovery in bothsublethally and lethally irradiated mice, even when theglucan was administered after irradiation. Glucan therapyin a mouse hind limb crush injury model caused a sig-nificant increase in boDe marrow proliferation whencompared to placebo therapy.14 While boDe marrow pro-liferation was not assayed directly in the present study, itis of interest that the glucan patients in the first week aftertrauma had consistently greater leukocyte counts than theplacebo group, although the differences were not statis-tically significant. Ready availability of peripheral leu-kocytes obviously would be a positive factor in resistanceto development of sepsis.

Glucan has been shown to exert many, ifnot all, ofitsprotective effects vio macrophage participation.12-14 Theprecise mechanism by which glucan activates macro-phages is poorly understood. However Czop and Usten58and Goldman59 have demonstrated the presence of specificbeta- 1, 3-glucan receptors on human macrophages andmast cell liDes. Activation of the befa glucan receptorsmay be the signal that initiates the cascade ofbiochemicalevents that culminate in macrophage activation. Studiesare underway to better delineate this glucan receptormechanism.

In summary to assess the use ofbiologic response mod-

MACROPHAGE ACfIVATION IN TRAUMAVol. 211 oNo. S 611.ifiers that enhance macrophage function, glucan, a beta-1, 3 polyglucose was used in a prospective, double-blind,randomized study oftrauma patients. While glucan ther-apy did not significantly decrease mortality rates due tosepsis, the overall rate of sepsis was less in the glucan-treated patients. Glucan-treated patients had a signifi-cantly increased serum IL-l level on day 3 after trauma.However by day 5 and thereafter, there was no differencein serum IL-I levels between groups. This early increasein IL-I correlated with subsequent skin test conversionfrom anergy to a positive response. Glucan therapy haQno effect on serum TNF levels. Furthennore neither serumIL-I nor serum TNF levels were predictive of subsequentseptic complications. Based on these preliminary results,we believe further clinical trials with BRMs that enhancemacrophage function are indicated in trauma patients.

ment by tumor necrosis factor-alpha improves antibiotic efficacyafter hemorrhagic shock. J Trauma 1989; 29:967-971.

17. Livingston DH, Appel SH, Sonnenfeld G, et al. Tbe effect oftumornecrosis factor-alpha and interferon-gamma on neutrophil func-tion. J Surg Res 1989; 46:322-326.

18. Sheppard BC, Norton JA. Cytokine protection against tumor necrosisfactor lethality. Surg Forum 1989; 40:80-82.

19. Duvic M, Reisman M, Finley Y, et al. Glucan-induced keratodermain acquired immunodeficiency syndrome. Arch Dermatol 1987;123:751-756.

20. Baker SP, O'Neill B. The Injury Severity Score: an update. J Trauma1976; 16:882-885.

21. Baker SP, O'Neill B, Haddon W, et al. The Injury Severity Score: amethod for describing patients with multiple injuries in evaluatingemergency careo J Trauma 1974; 14:187-196.

22. Moore EE, Dunn EL, Moore JB, et al. Penetrating AbdominalTrauma Index. J Trauma 1981; 21:439-445.

23. Kniker WT, Anderson CT, RoumiantzeffM. The Multitest system:a standardized approach to evaluation of delayed hypersensitivityin cell mediated immunity. Ann A11ergy 1979; 43:73-79.

24. Farina EC, Balbo G. ImmunologicaJ evaluation ofinfective risk inelective surgery. Surg Immunol1989; 2:21-34.

25. DiLuzio NR, McNamee R, Browder IW, et al. Glucan: inhibitionoftumor growth and enhancement ofsurvival in four syngeneicmurine tumor models. Cancer Treat Rep 1978; 62:1857-1866.

26. Mil1er SE, Mil1er CL, Trunkey DD. The imrnune consequences oftrauma. Surg Clin North Am 1982; 62:167-181.

27. Christou NY, Meakins JL. Neutrophil function in surgical patients:two inhibitors of granulOC)1e chemotaxis associated with sepsis.J Surg Res 1979; 26:355-364.

28. Alexander JW, Stinnett JD, Ogel CK, et al. A comparison of im-munologic profiles and their influence on bacteremia in surgica1patients with a high risk ofinfection. Surgery 1979; 86:94-104.

29. Saba TM. Reticuloendothe1ial systemic host defense after surgeryand traumatic shock. Circ Shock 1975; 2:91-107.

30. Scovill W A, Saba TM, Kaplan JE, et al. Deficits in reticuloendothelialhumoral control mechanisrns in patients after trauma. J Trauma1976; 16:898-904.

31. Hoffman MK. Macrophages and T -cells control distinct phases ofB-cell differentiation in the numoraJ imrnune response in vitro.J Immunol1980; 125:2076-2081.

32. UnanueER, Rosenthal AS. Macrophage Regulation ofIrnrnunity.New York: Academic Press, 1980.

33. Faist E, Mewes A, Baker CC, et al. Prostaglandin E2 (PGE2) depen-dent suppression ofinterleukin alpha (IL-2) production in patientswith major trauma. J Trauma 1987; 27:837-848.

34. Miller-Graziano CL, Fink M, Wu JY, et al. Mechanisms ofa1teredmonocyte prostaglandin E2 production in severely injured pa-tients. Arch Surg 1988; 123:293-299.

35. Hansbrough JF, Zapata-Sirvent RL, Shackford SR, et al. Irnrnu-nomoqulating drugs increase resistance against sepsis in trau-matized mire. J Trauma 1986; 26:625-629.

36. Hansbrough J, Peterson Y, Kortz EL. Post-bufO immunosuppressionin an animal model: monocyte dysfunction induced by burnedtissue. Surgery 1982; 93:415-423.

37. Williams DL, DiLuzio NR. Immunopharmacologic modificationof experimental viral diseases by glucan. EOS J Immunol Im-munopharmacol 1985; 5:78-82.

38. Williams DL, Cook JA, Hoffmann EO, et al. Protective effect ofglucan in experimentally induced candidiasis. J ReticuloendothelSoc 1978; 23:479-490.

39. Williams DL, Sherwood ER, Browder IW, et al. Pre-clinical safetyevaluation ofsoluble glucan. Int J Immunopharmacol1988; 10:405-411.

40. Dinarello CA. Interleukin-1 and the pathogenesis ofthe acute phaseresponse. N Engl J Med 1984; 311:1413-1418.

41. Kampschmidt RF. Leukocytic endogenous mediatorjendogenouspyrogen. In Pyanda MC, Canonico PG, eds. The Physiologic andMetabolic Responses ofthe Host to Infection and Inflammation.Amsterdam: ElsevierjN. Holland, 1981. pp. 55-74.

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investigators have suggested that la allows us to separate or differentiatefaci1itory macrophages from inhibitory macrophages, and this might alIowyou to stratif)' responsiveness or at least understand future clinical studies.

Finally, I have a question arising out ofignorance. Because glucan isa breakdo\\-n or one afilie components ofzymosan, and given the ma-terial that Dr. Deitch showed us earlier, did you see any gut-relatedcomplications in these patients?

DR. JORGE RODRIGUEZ (Buffalo, New York): This paper is a pro-spective, double-blind, randomized study. It clearly demonstrates that abiologic response modifier, glucan, increases macrophage monokineproduction in association with conversion for skin testing and a decreasein the overall septic response or rateo

My Questions are centered on the study design, the study population,and associated macrophage function.

The criteria for the inclusion ofthe study appears to be based solelyon operative procedure. Was there any attempt to develop an inclusioncriteria based on the level of severity of injury or contamination at thetime of injuf)1

Did the use of prophylactic anbibiotics, as mentioned by Dr. Baker,for 5 to 7 days without certification for contamination influence yourrate of septic complication?

Did the use of mechanical venti!ation on all postoperative patientsinfluence your pulmonary infection raje?

Thirty-eight per cent ofthe placebo group, with the higher septic rate,versus 6 percent ofthe study population, with the lower septic rate, wereinvolved in penetrating injury. Could ibis, in fact, account for the dif-ference in the septic rate?

Did the increase in macrophage production of monokine correlatewith any measure of antigen-processing presentation or a decrease inPGE-2 production?

My last Question concems the minimal increase in serum T:--"F in bothgroups. Could this be correlated to the time of specimen collection?

DISCUSSJON

DR. CHRISTOPHER BAKER (Chapel HiII, North Carolina): Dr. Browderhas contributed extensively to the literature on glucans, the polysacharidecomponent of zymosan, as an immunomodulator, and is the major, ifnot the only, surgicaI contributor in this area studying pancreatitis, woundhealing, and peritonitis in the last few yea~.

Much of the clinical work on immunomodulato~ in humans hasbeen done in Europe for various reasons; for instance, thymopentin inBelgium, NSAIDS in Germany "ith Faist (a collaborator ofmine), andnow Dr. Browder's study in ltaiy. Based on the work presented and thematerial in the manuscript, I have some methodologic questions and acouple comments.

Fi~t, how did you select the patients? One of the problems in thesestudies has aIways been trying to stratif)' the patients at risk, and I wonderif you could elaborate a little bit more.

I was interested in the use ofprophylactic antibiotics for 5 days becausethat is generally not my practice. Could you comment on that?

Can you comment on how glucan convens the anergic patients? Skjntesting is obviously a very complex afea. I notice that you had a largenumber of negative patients the fi~t day after operation, which I aIwaysthought was due to edema, but nobody has ever written much about it.In the data that you showed here this afternoon, you mentioned thecorrelation of IL-I and conve~ion on dar 3, but the other data for skjntest conve~ions were on dar 7 when the IL-I levels were not different.Perhaps you can comment on that,

Were the four abdominal abscesses in the placebo group due to laterecognition of injuries or were they abscesses after an operation for the

injury?As a methodologic point, you expressed the IL-I data as a percentage

ofthe dar 1 levels, which is a technique that we had to use a number ofyea~ ago because patients have a "ide variation between individuals interms of the level of response. Did you see this in terms of the rawnumbe~, and was that why you expressed the data as percentage? Ifthisis so, why did you express the TNF data in picograms instead of as apercentage? It mar be that the TNF data would also be significant iftheywere analyzed in the same way. We have usually followed patients inhuman studies for 3 weeks, and I wondered why you only followed themfor 10 days,

I wonder if you thought about measuring the presence or absence ofla antigen on the surface of these macrophages? As you know, other~

DR. WILLIAM BROWDER (Oosing discussion): Dr. Baker brought upthe selection of patients. Because of the requirernents for proper consentand Hurnan Use Cornrnittee approval, we had to exclude the patients IJisted on the slide, narnely, those with pre-existing renal or hepatic diseaseand patients with severe head injury.

Serious head injury seerned to be the injury that disqualified the rnost

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Nick DiLuzio, my late mentor in New Orlean's, was proud of the factthat he was the first to show that glucan was the pure polYglucose elementof zymosan, that is, zymosan devoid of the protein moieties and otherimpurities. He believed very strongly that they were two different com-pounds, with glucan possessing the immunostimulatory properties.

We have not seen sensitivity to endotoxin with the soluble glucan.We have not seen the inflammatory changes that are so common withzymosan.

Dr. Rodriguez, we did not objectify or quantitate the amount of con-tamination during operation in these patients. [t is of interest that theglucan group had a total of si¡¡ patients with colon injuries, whereas theplacero group had only two patients with colon injuries. AII colon injurieswere managed with colostomy. Failure to quantitate contamination is aweak point in the study, but on the surface it appears that the two groupswere comparable.

The mechanicaI ventilator is a difficult paramenter to quantitate.Overall it is very difficult to study patient groups that are 'similar' aftera traumatic insulto In the present study we individualized mechanicalventilation management in these patients and weaned them offthe ven-tilators as rapidly as they could tolerate. It is interesting that the threepatients who developed pneumonias carne offthe ventilator quite rapidly,and we could not relate the pneumonia to prolonged ventilation.

We did not look at PGE-2 levels in this particular study. We havedone many animal studies, such as one using a hind limb crush modelin which glucan stimulation promptly increased IL-I release while actuaIlydecreasing PGE-2, so there appears to be some differentiaI in the responseof the macrophage in this setting.

1 would agree with Dr. Rodriguez that theTNF levels might be timerelated in the sense that by measuring them at inappropriate times ornot frequently enough one might certainly miss a very transient increase,and these studies simply have to be extended and aSsayed in more detail.

Let me quote a Chinese proverb: 'A joumey of a thousand miles beginswith a single step.' What we are seeing with this study and other clinicalstudies that are evaluating biologic response modifiers are those first fewsteps in that long joumey. I hope it will end with new benefits to thetrauma patient.

patients, because this trauma center sees quite a number of multiplyinjured patients. In addition, we tried to restrict the study to patientswho underwent operative intervention, again trying to document a specificinjury as opposed to blunt trauma or pelvic fracture patients who didnot undergo operative intervention. In these Jatter patients we wouldnever be able to specify the exact extent ofthe injury.

The 5 days of antibiotics caused some concern. This is the standardpolicy at the Trauma Institute where the study was carried out. At leastI can say that it was consistent throughout both groups of patients.

Dr. Baker asked about the raje of the macrophage in the skin testconversion. Unfortunately we did not look specifically at that. In thisstudy we might hypothesize that macrophage antigen processing andpresentation would be an essential aspect of proper lymphoctye res'ponseand positive skin test reactivity. There have been many studies showingthat this macrophage antigen processing ability is lost with hemorrhageand hemorrhagic shock and trauma. Thus this is a possibility, althoughwe have no direct evidence in this study.

AII of these abdominal absoesses followed operative intervention. Therewere two after duodenal injuries, one after hepatic injury, and one aftera bowel injury.

Dr. Baker brought up the point about expressing IL-I values as apercentage increase, and he was correcto Because of the wide variabilityof ILol in these patients, it is very difficult to get any meaningful rawnumbers.

The same was true for the serum TNF levels. If you looked at thepercentage increase in that group, they showed no difference betweenthe glucan or placebo groups.

The IO-day limit on the study, as opposed to 3 or 4 weeks, was de-termined simply because of availablity of the radioimmunoassay. Cer-tainly the next step in this area ofinvestigation would be to follow thesepatients longer as they develop septic complications, and to look at whatimmune parameters are disrupted at that time.

The lA antigen on the macrophage was mentioned. We did not studythis factor. Again, we preferred to use interleukinol as the marker forreflecting facilitory macrophages or macrophage activation.

The question ofzymosan versus glucan is an intersting one. Historically,

I