Immediate Burn Wound Excision Restores AntibodySynthesis to Bacterial Antigen

HIROMASA YAMAMOTO, M.D., SORANIT SILTHARM, M.D., SUZAN DESERRES, B.A.,C. SCOTT HULTMAN, M.D., AND ANTHONY A MEYER, M.D., PH.D.

Department of Surgery and North Carolina Jaycee Burn Center, University of North CarolinaSchool of Medicine, Chapel Hill, North Carolina 27599.7210

Presented at the Annual Meeting ofthe Association fo!' Academic Surgery, Dearborn, Michigan, November 8-11, 1995

Although burn wound excision and grafting havebeen shown to improve patient survival, the effects onimmune function, especially humoral immunity, arenot completely understood. The purpose of this studywas to investigate the effect of immediate and earlywound excision on antibody synthesis and B-cell pro-liferation, specifically, antibody response to PGPS, aubiquitous bacterial cell wall antigen. Thirty-six maleBALB/c mice were divided into tour groups. Sham micereceived no burn, and remaining mice received a 30%body surface ares full-thickness bum. Under generalanesthesia, excision and grafting was performed ei-ther 6 or 72 hr after injury (BE&G6 and BE&G72groups). A fourth control group received bum but didnot undergo excision and grafting (Burn group).Splenocytes were isolated 8 days postbum and stimu-lated with 2.5 JLg/ml lipopolysaccharide. Anti-PGPSIgM, total IgM, and total IgG levels were determinedby ELISA. B-cell proliferation, measured by (3H]-thymidine uptake, was expressed as stimulation index.All B-cell functions were significantly suppressed byburn, injury. Immediate excision and grafting (BE&G6)restored anti-PGPS IgM synthesis to normal, whilenonspecific B-cell functions did not change signifi-cantly. Early excision and grafting (BE&G72), how-ever, failed to significantly improve any B-cell func-tions. Immediate but not early BE&G restored anti-body synthesis to the bacterial cell wall antigen(PGPS). Immediate BE&G may therefore lead to a de-crease in bacterial infection after burn injury. (t) 1996Academic Press, Inc.

INTRODUCTION

Severe thermal injury results in host immunosup-pression, impairing resistance to infection and produc-ing significant morbidity and mortality. Many alter-ations in the immune system have been studied, includ-ing humoral immunity and cell-mediated immunity.Impaired cell-mediated immunity, such as suppresseddelayed-type hypersensitivity [1], prolonged survival ofallograft skin [2,3], altered T-cell blastogenesis [4-6],

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and decreased IL-2 production [7, 8] have been re-ported. Our laboratory has investigated altered hu-moral immunity, specifically B-cell function. We havepreviously demonstrated that both specific and nonspe-cific antibody synthesis were suppressed at 8 days post-burn [9]. Schlüter et al. alBo reported impaired activa-tion, proliferation, and differentiation process ofB-cells[10]. The goal of our research is to investigate the mech-anism ofthis specific and nonspecific B-cell suppressionafter burn injury. In this experiment, we focused onthe role of the burn wound in B-cell suppression.

Early burn wound excision and grafting, first pro-posed by Young [11], was adapted to the care ofsevereburn patients by Meeker and Snyder [12] and MacMil-lan [13] with favorable results. This treatment has be-come the standard therapy supported by more recentstudies [14, 15] which have shown improved survival,decreased complications, and decreased hospital stays.Such therapy was alBo reported to be effective in chil-dren [16, 17].

The effects of excision and grafting on immune func-tion have alBo be en examined by laboratory investiga-tors, especially on cell-mediated immunity [18-23].Hultman et al. reported that burn wound excision re-stored suppressed cytotoxic T-Iymphocyte function[18]. Cetinkale et al. showed that cell-mediated immu-nity, as measured by popliteallymph no de assay, wasrestored by excision and grafting [19]. Hansbrough alBoexamined the cell-mediated immunity determined byear swelling following dinitrofluorobenzene (DNFB)sensitization. They showed that excision of the burnwound at 2 hr following injury resulted in restorationofimmunosuppression, but that escharectomy at 24 hrafter burn resulted in significant suppression of cell-mediated immunity [20]. Concerning the timing of exci-sion and grafting, Tchervenkov et al. reported similareffects of excision and grafting on neutrophil function[24]. Their study showed that neutrophil delivery to adelayed-type hypersensitivity reaction and a bacterialskin lesion was restored to normal if the burn woundwas removed 24 hr postburn. However, waiting 3 or 7days after burn trauma to excise the wound resultedin partial or no improvement in neutrophil delivery.

158 JOURNAL OF SURGICAL RESEARCH: VOL. 63, NO. 1, JUNE 1996

Because the effects ofburn wound excision on humoralimmunity have not been assessed, we investigatedthese effects on B-cell function. We hypothesized in thisstudy that B-cell function would be restored ifthe burnwound eschar was excised at a certain time point afterburn injury, but that it would not be restored by laterexcision.

In this study, we investigated the effects of burnwound excision on antibody synthesis and B-cell prolif-eration. For antibody synthesis, we measured antigen-specific antibody synthesis and polyclonal IgM and IgGsynthesis. Phipps et al. reported that specific and poly-clonal antibody production were regulated differently[25] and McLeish et al. showed different response toPGE2 between specific and nonspecific antibody syn-thesis [26]. We hypothesized in this experiment thatburn wound excision may be effective on some B-cellfunctions but not others. We chose to study antibodyresponse to peptidoglycan polysaccharide (PGPS).PGPS is a ubiquitous bacteria! antigen found in bothgram-positive and gram-negative bacteria [27], and hassimilar pathophysiologic effects to lipopolysaccharide(LPS), which is only present on the surface of gram-negative bacteria. Nonspecific B-cell function was ex-amined by B-cell proliferation in response to LPS, aswell as total IgM and IgG production.

METHODS

Animals. Twenty- to twenty-five-gram, male BALB/c mice (Har-lan Sprague-Dawley, Inc., Indianapolis, IN) were used in this exper-iment. AlI animal protocols were approved by the Committee on Ani-mal Research, University of North Caroli~a at Chapel Hill.

Experimental designo Thirty-six male BALB/c mice were random-ized to received either a 30% BSA full-thickness bum or a Shambum. Excision and grafting was performed either 6 or 72 hr afterbum (BE&G6 and BE&G72 groups). A fourth control group receivedburn injury but no excision (Bum group). Splenocytes were isolated8 days postburn and cultured with 2.5 JLg/ml LPS, a nonspecific B-cell stimulator. LPS is necessary to stimulate B-cell proliferation tothe point antibody production is detectable. We previously testedanti-PGPS antibody and found that it does not cross-react with LPS[28]. Anti-PGPS IgM, total IgM, and total IgG levels were measuredby ELISA. B-cell proliferation, measured by [3ffithymidine uptake,was expressed as stimulation index (SI).

Thermal injury. Under general anesthesia with meti1,oxyfiurane(Pitman-Moore, Washington Crossing, NJ), animals were circumfer-entially clipped. Three groups of bumed mice received a full-thick-ness contact bum by applying a 65-g copper red, previously heatedto 100°C, to the animal's back for 10 seco Four individual copperrod applications were required to create a'30% TBSA bum, as oneapplication represented 7.5% of fue animara TBSA. Animals wereresuscitated by injecting 2.5 mI oflactated Ringer's intraperitoneally,and a 3 mgikg dose ofmorphine sulfate (Eli Lilly and Co., Indianapo-lis, IN) was administered subcutaneously.

Burn wound excision and grafting. Animals in BE&G6 and BE&G72 groups had their burn wounds completely excised 6 and 72 hrpostburn, respectively, under methoxyfiurane anesthesia. Excisionwas performed with sharp dissection down to the level ofmusculofas-cia. Syngeneic full thickness skin from another BALB/c mouse wasgrafted to clase the wound defecto

Splenocyte cultures. Spleen cell suspension from each animal wasprepared as previously described [9]. Briefiy, all animals were sacri-ficed 8 days after injury, and spleens were removed and washed withcold Hanks' balanced salt solution with 2000 !U/mI penicillin and2000 JLg/ml streptomycin. They were cut in hall, the pulp was dis-

persed into the RPMI-1640 media supplemented with 5% heat-inacti-vated fetal bovine serum, 1 mmol/liter sodium pyruvate, 2 mmoVliter L-glutamine, 1% nonessential amino acids, 1000 IU/mI penicil-lin, 1000 JLg/ml streptomycin, and 40 JLmol/liter 2-mercaptoethanol(2-ME; Mallinckrodt, Inc., Paris, KY), and the membrane was dis-carded. AlI tissue culture materials were obtained from the TissueCulture Facility (Lineberger Cancer Research Center, University ofNorth Carolina-Chapel Hill), except for 2-ME. The red blood cellswere lysed by distilled water and splenocytes were resuspended. Theviability of lymphocytes determined by trypan blue exclusion wasconsistently greater than 98%. Spleen cell suspension was adjustedto a final concentration of 1.5 X 106 cells/ml.

One hundred microliters ofthe spleen cell suspension prepared abovewas cultured with 5.0 JLg/mI Escherichia coli 0111:B4 LPS (SigmaChemical Co., Sto Louis, MO) in each weIl of a 96-weIl, flat-bottomedmicrotiter culture dish (Costar Corp., Cambridge, MA). The final con-centration of LPS was 2.5 JLg/mI and fue final volume in each weIl was200 JLI. Culture plates were incubated at 3rC, 5% CO2.

B.cell proliferation. B-cell proliferation was measured using thesplenocyte culture described above. Platea were cultured at 37°C, 5%CO2 for 72 hr, and for the final 24 hr, 1 JLCi of tritiated thymidine(ICN Biomedicals, Irvine, CA) in 20 JLI of media was added to eachwell. Cells were harvested using a semiautomatic cell harvester (Ska-tron Inc., Sterling, VA) onto Xtalscint filters (Beckman Instruments,Columbia, MD). Filters were placed into scintiIlation vials andcounted on a Beckman LS6000TA scintillation counter.

ELISA for anti-PGPS IgM. Anti-PGPS IgM was measured byELISA as previously described [9]. Briefly, supernatants from thesplenocyte culture were collected after 5 days of incubation. Sampleswere stored at -80°C for use in assays. The 96-weIl EIA/RIA plates(Costar Corp., Cambridge MA) were coated with 2 JLg/ml PGPS in0.1 M carbonate buffer (pH 9.6) and incubated at room temperaturefor 2 hr. Unbound sites were blocked with 200 JLVwell Blotto (5%nonfat dry milk with 0.02% sodium azide in PBS) at 4°C overnight.Fifty microliters of undiluted tissue culture supernatants and stan-dards (serially diluted control mouse anti-PGPS serum) were addedto the platea. Plates were incubated at 4°C overnight and washed.Fifty microliters per weIl of alkaline phosphatase-labeled goat anti-mouse IgM was added to the plates and plates were incubated for 2hr at room temperature. The plates were washed and 50 JLVwell ofp-nitrophenyl phosphate in substrate buffer was added to the plates.After 30 min ofincubation in the dark, optical density was measuredat 405 nm on an automatic plate reader (EL311s Autoreader, Bio- TekInstruments, Inc., Winooski, VT). Nondiluted anti-PGPS standardserum from immunized BALB/c mice was arbitrarily set at 104 U/mI. Concentrations of anti-PGPS IgM in mU/ml for each specimenwere calculated from the standards.

ELISA for total IgM and IgG. Total IgM and IgG were measuredby ELISA as previously described [9]. Briefiy, the 96-weIl EIA/RIAplates (Costar Corp., Cambridge MA) were coated with 5 JLg/ml ofgoat anti-mouse IgM and IgG in 0.1 M carbonate buffer (pH 9.6) andincubated at 4°C overnight. Plates were blocked with 100 JLVwell ofPBS with 0.2% Tween 20 for an hour and washed. Fifty microlitersof standard IgM or IgG solution was diluted serially in PBS with 2%FBS, and diluted culture supernatants were added to the plates.After overnight incubation at 4°C, plates were washed and 50 JLVwell of alkaline phosphatase-labeled goat anti-mouse IgM or IgG (1JLg/ml solution, diluted in PBS with 0.02% Tween-20) was added tothe plates. After 2 hr of incubation at room temperature, the plateswere washed and 50 JLVwell ofp-nitrophenyl phosphate in substratebuffer was added. After 30 min of incubation in fue dark, opticaldensity was measured at 405 nm on an automatic plate reader(EL311s Autoreader; Bio-Tek Instruments, Inc.). Concentrations ofIgM or IgG for each specimen were calculated from the standards.

Statistical analysis. AlI data are expressed as means :!: the stan-dard error of the mean (SEM). Results were compared by Student'st test. Probabilities less than 0.05 were considered statistically sig-nificant.

RESULTS

Effects of BE&G on B-cell proliferation. B-cell pro-liferative response stimulated with LPS was measured.

YAMAMOTO ET AL.: IMMEDIATE EXCISION AND B-CELL FUNCTION 159

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FIG. 2. Effect of BE&G on anti-PGPS IgM synthesis in spIeno-cyte culture stimulated with LPS. BE&G6 and BE&G72 indicate theanimaIs received BE&G at 6 and at 72 hr postburn, respectiveIy. AlIthe vaIues are expressed as means :t SEM. *p < 0.05 compared withSham group animaIs. tP < 0.05 compared with Burn group animaIs.

SHAM BURN BE&G6 BE&G72

FIG. 1. Effect of BE&G on splenocyte proliferative response toLPS. BE&G6 and BE&G72 iridicate the animals received BE&G at6 and at 72 hr postburn, respectively. Al1 the values are expressedas SI (mean :t SEM). SI were calculated as the counts with LPSdivided by the counts with media. *p < 0.05 compared with Shamgroup animals.

specific IgM and IgG synthesis was not restored byimmediate (BE&G6) or early excision and grafting(BE&G72).

DISCUSSION

It is accepted that the immunosuppression of ther-mal injury is associated with sepsis, which remains amajor cause ofdeath after burn injury. Suppression ofcell-mediated immunity has been extensively studied,including altered distribution of T-cells [29, 30], de-creased response to mitogens [6], and decreased IL-2production [7, 8]. Suppressed delayed hypersensitivity[1], prolonged survival of allograft skin [2, 3], and sup-

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Burn injury induced a significant suppression in B.cellproliferation compared with Sham' group animals (31.4:!: 2.29 in Burn group vs 58.4 :!: 3.51 in Sham group).lmmediate (BE&G6) and early excision and grafting(BE&G72) did not change B-cell proliferation signifi-cantly, compared to Burn group. B-cell proliferation inthese groups was significantly suppressed comparedwith Sham group animals. These data are shown inFig.1.

Effects of BE&G on anti-PGPS IgM. Specific anti-body synthesis was assessed by measuring anti-PGPSIgM synthesis. Anti-PGPS synthesis in Burn group wassignificantly suppressed compared to Sham group (187:!: 12.9 U/mI in Sham group vs 98.5 :!: 10.0 U/mI inBurn group). lmmediate excision (BE&G6) signifi-cantly increased anti-PGPS IgM synthesis comparedto Burn group (180.8 :!: 42.7 U/mI, P < 0.05 comparedwith Burn) and there was no statistical difference be-tween BE&G6 and Sham group (P > 0.05 comparedwith Sham). Delay in excision and grafting to 72 hrpostburn (BE&G72) resulted in no improvement inanti-PGPS synthesis (97.9 :!: 9.24 U/mI in BE&G72, P< 0.05 compared with BE&G6). There was no signifi-cant difference between BE&G72 group and Burngroup. These data are shown in Fig. 2.

Effects of BE&G on total IgM and IgG. The effectsof BE&G on nonspecific IgM and IgG synthesis areshown in Figs. 3 and 4, respectively. Again burn injuryinduced significant suppression in nonspecific immuno-globulin synthesis compared to Sham animals. TotalIgM synthesis in Burn group animals was 17.3 :!: 0.72j.lg/ml and was significantly lower than Sham group(38.0 :!: 2.96 j.lg/ml). Total IgG synthesis in Burn groupwas 0.618 :!: 0.058 j.lg/ml and was significantly lowerthan Sham group (1.127 :!: 0.165 j.lg/ml). Unlike anti-gen-specific antibody synthesis, the suppressed non-

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SHAM BURN BE&G6 BE&G72

FIG.3. Effect ofBE&G on total IgM synthesis in splenocyte cul-ture stimulated with LPS. BE&G6 and BE&G72 indicate the animalsreceived BE&G at 6 and at 72 hr postburn, respectively. AlI thevalues are expressed as means = SEM. *p < 0.05 compared withSham group animals.

160 JOURNAL OF SURGICAL RESEARCH: VOL. 63, NO. 1, JUNE 1996

-SHAM BURN BE&G6 BE&G72

FIG.4. Effect ofBE&G on total IgG synthesis in splenocyte cul-ture stimulated with LPS. BE&G6 and BE&G72 indicate the animalsreceived BE&G at 6 and at 72 hr postbum, respectively. AlI thevalues are expressed as means :t SEM. *p < 0.05 compared withSham group animals.

sion which was measured by DNFB sensitization, T-cell proliferation, and neutrophil oxidative burst [37,38]. Ferrara, Dyess, and their colleagues reported thatsubeschar wound Huid acted as a reservoir for systemicimmunosuppressive factors [39, 40]. Sparkes et al.stressed the presence of cutaneous burn toxin andshowed that this substance may be responsible for im-munosuppression [41-43].

Clinically, most investigators still believe that onlyremoval of burn eschar can correct the host immunefunction. Early burn wound excision was first proposedby Young [11] and adopted by Meeker and Snyder [12]and MacMillan [13] in the 1950s. However, the timingand extent of excision and grafting remained controver-sial. Immediate eschar excision and wound closure be-carne popular in the 1970s and Burke et al. reporteddecreased morbidity and mortality as well as decreasedhospital stay [14]. Tompkins et al. algo [15] showed intheir retrospective study on over 1100 patients thatsurvival was best in 1979 through 1984 when earlywound excision became the standard of therapy, withno significant change in fue distribution of burn size,age, or sexo Beneficial effects of early excision on chil-dren [16, 17] and patients without inhalation injury[44] have algo be en reported.

In addition to clinical data demonstrating the advan-tage of early excision and grafting, many laboratoryinvestigators have algO shown improvements by woundexcision in immune function. Hansbrough et al. exam-ined the effect of early wound excision and primaryclosure on cell-mediated immunity, as measured by earswelling following DNFB sensitization [20]. Theirstudy showed that immediate wound excision and pri-mary closure restored suppressedcell-mediated immu-nity. Cetinkale et al. reported the restoration of cell-mediated immunity measured by popliteallymph nodeassay, which assesses lymph node response to allogenicsplenocytes [19]. However, reports using a murinemodel showed excision and grafting with syngeneicskin was itself suppressive in cell-mediated immunityand that the suppression was additive to that of burninjury [22, 23]. Concerning humoral immunity, the ef-fects of early excision and grafting have not been exten-sively investigated.

In this experiment, B-cell function, as determinedby proliferation assay and antibody production, wasimpaired 8 days after burn injury. These results wereconsistent with our previous studies [9, 45] and withthe results of Schlüter et al., who observed that B-cellproliferation and immunoglobulin synthesis were im-paired by the 2nd week after burn injury [10]. We stud-ied B-cell function on Postburn Day 8, not only becauseour previous studies showed a significant suppressionin immunoglobulin synthesis but algo because it wasa time point at which various immune functions aresuppressed. Deitch et al. examined the time course oflymphocyte response to mitogens [6] and showed thatsuppression of lymphocyte proliferation stimulated byphytohemagglutinin, concanavalin A, and pokeweedmitogenwas maximal 5-7 days after thermal injury.

pressed cytotoxic T -cell function [3] have been algo re-ported. Although antibody response is important in re-sistance to bacteria, a limited number of studies havebeen done on B-cell responsiveness. We have previouslydemonstrated suppressed specific and nonspecific anti-body synthesis after burn injury [9]. Our laboratoryhas focused on specific antibody synthesis to PGPS,which is the principal component of bacterial cell walland is algo a ubiquitous bacterial antigen found in bothgram-positive and gram-negative bacteria [27]. Anti-body response to LPS has been measured as a primaryresponse to bacteria, but LPS is present only on thesurface of gram-negative bacteria. PGPS algo haspathophysiologic effects similar to LPS, including acti-vation of TNF. Antibody response to PGPS has beeninvestigated in Borne diseases [31-33], but the re-sponse after burn injury has never been examined butby our laboratory. Our previous studies have shownthat anti-PGPS antibody synthesis is increased 24 hrafter burn injury, and bacterial translocatioñ occurs atthe same time [9, 34]. Total immunoglobulin synthesis,however, remains normal 24 hr postburn. We believethat this specific antibody response may be a primaryhost response to bacteria. When the first impact ofbac-terial challenge is cleared on Postburn Day 3, anti-PGPS antibody production returns .to normal. On Post-burn Day 8, however, both specifié and total immuno-globulin synthesis are suppressed.

A number of investigators have attempted to clarifythe mechanism of defects in cell-mediated and humoralimmunity by measuring the production of cytokine [7,8] or by administering immunomodulators includingindomethacin and IL-2 [35,36]. However, many studieshave shown that burn wound plays an important Tolein immunosuppression. Hansbrough et al. showed thatburned murine skin, when transferred onto an unin-jured animal, can produce systemic immune suppres-.

YAMAMOTO ET AL.: IMMEDIATE EXCISION AND B-CELL FUNCTION 161

skin or subeschar fluido PGE2 levels were observed tobe increased in burn wound skin [46].

In conclusion, we demonstrate that burn injury in-hibits B-cell proliferation, specific antibody production,and nonspecific antibody production. We algo demon-strate that immediate burn wound excision restoresantibody synthesis to bacterial antigen, but does notrestore nonspecific B-cell response. Delaying excisionand grafting to 3 days postburn results in no improve-ment in suppressed B-cell function. Involvement ofPGE2 as a factor from burn wound was implicated.Immediate excision and grafting may lead to a decreaseof bacterial infection after burn injury.

REFEREN CES

Moss et al. reported maximal suppression (68%) of IL-2 production at 7 days [7]. Some authors stressed thatthe presence of activated suppressor T-cells followingthermal injury may be responsible for immunosuppres-sion. McIrvine et al. showed that the inversion of thenormal ratio between suppressor/cytotoxic and helper/inducer subsets occurred soon after burn injury,reached a peak in 5 to 7 days, and then returned gradu-ally to normallevels by 14 days [29]. Antonacci et al.algo showed that the ratio between CD 4 and CD 8 wasminimal ón Postburn Day 7 [30].

In the present study, we demonstrated that irnmedi-ate excision and grafting (6 hr postburn) restored anti-PGPS IgM synthesis to baseline controls. However, adelay in wound excision to 72 hr resulted in no improve-mento Similar phenomena concerning the timing ofBE&G were reported by Tchervenkov et al., who mea-sured neutrophil delivery to a delayed-type hypersensi-tivity reaction and a bacterial skin lesion [24]. Neutro-phil delivery suppressed by burn injury was restoredto normal by excision and grafting 1 day after burninjury, but waiting 3 or 7 days to excise the woundresulted in partial or no restoration ofneutrophil deliv-ery. Hansbrough et al. algo examined the timing ofwound excision [20]. They demonstrated that excisionof the burn wound 2 hr after burn restored cell-medi-ated immunity, but escharectomy 24 hr postburn re-sulted in significant immunosuppression. Our presentstudy and these two studies indicate that only veryearly burn wound excision can restore systemic im-mune function. It is, therefore, suggested that burnwound or Borne substance from burn wound may con-tribute to the systemic immune response.

We algo showed in this experiment that nonspecificB-cell functions, such as total IgM synthesis, total IgGsynthesis, and B-cell proliferation, were not changed byimmediate or early excision and grafting. These resultssuggest that specific and nonspecific antibody synthesisare regulated differently. In a previous study, we founda similar phenomenon concerning the difference be-tween specific and nonspecific antibody response [45].We used the same burn model and sacrificed the ani-mals 8 days postburn. We examined the effects of exog-enous indomethacin, cyclooxygenase inhibitor. Indo-methacin restored anti-PGPS IgM to normal, but B-cell proliferation and nonspecific antibody synthesisremained suppressed. Phipps et al. reported that mac-rophages strongly inhibit antigen-specific B-cell re-sponsiveness by two signals, antigen-antibody com-plex and prostaglandin E2 (PGE2). They algo showedthat polyclonal antibody synthesis was inhibited byPGE2 but that the mechanism of this inhibition wasdifferent from the antigen-specific one [25]. McLeish etal. algo reported the different response to PGE2 be-tween specific and nonspecific antibody synthesis [26].From these results, it was suggested that PGE2 maybe a factor which comes from burn wound and impairssystemic immune function. Hansbrough et al. and fer-rara et al. algo suggested that PGE2 may be responsiblefor the immune suppression caused by burn wound

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