Perioperative Recombinant Human Granulocyte Colony- Stimulating Factor (Filgrastim) Treatment Preven ts Immunoinflammatory D ysfunction Associated With
Major Surgery
Christian Schneider, MD,
*
Sonja von Aulock, MD,t
Siegfried Zedler, PhD,*
Christian Schinkel, MD,*
Thomas
Hartung,
MD, PhD,*
and EugenFaist,
MD*Objective: To examine theeffects of perioperati ve rhG-CSF admin- istration onimmune function in patients subjected to majorsurgery,
Summary Background Data: Severetrauma,such asmajorsur- gery,initiatesacuteimmunodysfunctionwhich predisposesthe pa- tienttowards infectious complications.
Methods: Sixtypatientsundergoing elective surgery receivedeither recombinant human granulocyte colony-stimulating factor/rh G- CSF (Filgrastim)or a placebo perioperatively,At severaltimepoints before andafter thesurgical intervention immunofunctional param- eterswere assessed.
Results: Leukocyte counts and serum levels of anti-infl ammatory mediators (IL-lra and TNF-R) were increasedin Filgrastim-treated patients,whilethepost-operative acute phase response was attenu- ated.Monocyte deactivation (reducedTNF-11releaseand HLA-DR expression)and lymphocyte anergy(impaired mitogenicprolifera- tion and reduced TI-Il lymphokinerelease) wereblunted and the incidence and severityofinfectiouscomplications were reduced. Conclusions: These resultssuggest thatFilgrastim treatmentrein- forces innate immunity, enabling better prevention of infection.
Thus, thisuniquecombination of hematopoietic,anti-inflammatory and anti-infectiouseffects on the innate immunesystem warrants further studyof clinicalefficacyand sepsisprophylaxis.
(AnnSurg 2004;239:75-81)
M
aj or surgery carries an increased risk of infectious complications.The body reactssystemically to trauma by the acute phase reaction.Localtrauma and inflammationFrom *K linikul11 Grosshadern, Ludwig-Mnxlmilinns- University, Munich, Germany; and [Biochemical Pha rmacology, University of Konstanz, Konstanz,Germany ,
Reprints:Eugen Faist,MD, FACS, Departmentof Surgery, Ludwlg-Maxi- milians-University, Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich ,Germany,E-mail: Faist@gch.med.uni-n1Ucnchen.dc.
are accompanied by anti-Inflammatory counterregulation, which leaves immune cells in a state of anergy, impairing host defense against infection.I Post-traumatic immunopa- ralysis involves both monocytes and lyrnphocytes and is reviewed in detail elsewhere.' The capacity of monocytes to react toaninflammatory stimulus byreleasingtl11110r necrosis factor(TNF)-a is diminished.'This correlateswith decreased expression of HLA-DR,a major histocompatibility complex subtype, on their surface." Lymphocytes have a reduced capacityto produceTill typecytokines, interleukin-2 (IL-2) and interferon-y(IFN-y), and are inhibited in their ability to proliferate't"while production ofTr-r2 type cytokines such as IL-4 isup-regulated."Prevention of post-traumatic immuno- paralysisand reduction of therisk of trauma-induced infec- tion could be brought about by strengthening and priming hostdefense before trauma, improving the immune system's capacity to cope with invading pathogens locally and pre- venting into a systemic anti-inflammatory response.
The granulocyte colony-stimulating factor (G-CSF) playsa central rolein the endogenous response to infection and inflammation.X G-CSF stimulates hematopoiesis and primes granulocytesfor enhanced immune defense, On the other hand,therelease ofproinflammatorymediators such as TNF-a, IL-I/3, and IL-12 by monocytes in response to an infectious stimulus is reduced by G-CSF, while these cells are primed toward all increased release of anti-inflammatory mediatorssuch as IL-lra. sTNF- receptors p55 andp75.'J·1O Therefore, G-CSF seems to be a promising candidate to signalthe immune system to prepare for defense, while not activatingimmunecells directly,and to limitthe inflamma- tory response by attenuating monocytes,allowing 1110re mod- erate reactions but not paralyzlng these regulators.
Datafrom preclinical studies support this hypothesis:
Faster recruitment of neutrophils to the site of infection,I1 reduced spreading of bacteria12.13 and better survival of endotoxin-inducedhepatotoxicity and shockl4,rs as well as
75
First publ. in: Annals of surgery 239 (2004), 1, pp. 75-81
Konstanzer Online-Publikations-System (KOPS)
Post-operative cours e
multimicrobial sepsis16-t8havebeen reportedin rodentmod- els inwhichG-CSFwasadministeredbefore or with trauma.
Improved cardiovascularfunction, endotoxin clearance, and survival withG-CSFtreatmentwere also reported in ca nine models of septic shock.19•20 Only a few human trials of G-CSFassepsis prophylaxis have been carriedout. G-CSF treatment significantlyreduced infectious complications in 19 cancerpatients undergoing esophagectomycompared with 77 control patients." G-CSF treatment reduced theincidenc e of multipleorgan failure in756 pneumoniapatients22as well as in 37 liver allograft recipients.f
The evidence from the above-descri bed animal and limited clinical studies suggested to us that prophylactic treatment with G-CSF at thetime a risk canbe anticipated, such as before an operation, may offer protection from infections and lower the incidence of sepsis. Thus. wi th this original study,we wanted toinvestigate the impactof continu- ousperioperativerhG-CSFadministrationonpostoperative im- munoinflarnmatory function as wellas the incidence and out- come of infections inpatients havinghigh-risksurgery.
MATERIALS AND METHODS
Sixty patients were included in a randomized,placebo- controlled,double-blindstudythatwas performedaccording tothe requirements of the Helsinki declaration, The patients either received placebo 01'5 [.kg recombinant human granu- locyte colony-stimulating factor/rh G-CSF (Filgrastim)/kg s.c. on days -2, 1,and3relative to surgery (Group A) or 5 [.kg/kg on day - 2 and 2 [.kg/kg on eachof the 5 subsequent days (GroupB). The total close receivedwas15 [.kg Filg ras- tim/kg bodyweightin bothgroups(Fig. I).
The study design was approvedbythe ethical review board of theLudwig-Maximilians-UniversityMunich.Exclu-
Prospcctlvc
raudotnlzaticn Surgery
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day -2 -1 0 1 2 3 5 7
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Group B 5jJg 2jJg 2jJg 2jJg 2jJg 2jJg
Immune-
* * * *
*monitoring
FIGURE1. The experimentalprotocolof the studyisdepicted.
Patients in thetreatmentgroup received rhG-CSF(15 j-tg/kg body weight) over 6 perioperatlve days either as 3 bolus administrations of5 /lg/kg bodyweight or as acon ti n uou s administration of 2 /lg/kg body weight following an init ial bolus of5J..I.g/ kg bodyweight.
sion criteria were pregnancy, immunosuppression (chemo- therapy,steroid medication, AIDS, etc.), acuteinflammatory diseases (indicated by increased C-reactive protein serum levels),andpreexistingrenal failurerequiring hemodialysis.
The underlying diseases for all included patients wereasso- ciatedwithmalignancy. Allpatients gave informed consent.
No side effectsof subcutaneous Filgrastim administration were observed.
The types of surgery performed in the placebo versus Filgrastim groupand otherdemographic data are depictedin Table I.As it hasbeen documented, the groups did not differ significantlyin terms of age, gender distribution,and magni- tude of surgical trauma as expressed through the length of operationtime (4.3 :t 0.4 hours in the placebo vs. 4.6 ± 0.2 hours in the Filgrastim group).
Whole Blood Incubation
Blood samples forimmunomonitoring were withdrawn on days-2,1,3, 5, and 7. Differential white blood cell counts were assessed at everytime of blood collection.Whole blood incubations were performed in sterile Vacutainers (Sarstedt, Ninnbrecht,Germanyi4•25;5mL20% whole bloodinRPMI
TABLE 1. Patient Demographics
rhG-CSF Control Group A1B (n =20) (n= 40)
Sex (M:F) 14:6 30:[0
Age (years ± SEM) 61 ± 2 66± 3
Type ofsurgery
Largebowel resection 10 15
Gastrectomy 4 9
Whippleprocedure 1 3
Esophageal resection with 2 4
colon interposition
Partial hepatectomy 2 6
Anatomic lung resection 1 3
Operationtime (hours± SEM) 4.3± 0,4 4.6± 0,2 Loss of blood during Surgery 1078 ± 164 1650± 266
(mL ± SEM)
Units of packed RBCs 2,9± 1,5 2,2±0,6 administered
Length of stay at the ICU 156± 103 64± 26 (hours± SEM)
Controlled ventilation at the 24± 18 29± 14 ICU (hours± SEM)
Length of stay at the hospital 31 ± 5 26±2 (days ± SEM)
Sixtypatientsundergoingmajorsurgicalprocedureswere enrolled and randorni zedtoreceive either rhG-CSF or placebo.Age,sexdistribution.and qualityofsurgeryare documented.Fornone ofthe factors were significant differences found between both groups,RBes.red blood cells.
1640 (BioWhittaker, Verviers, Belgium) supplemented with 100 IV penicillin, 100 /Lg/mL streptomycin and 2.5 IV heparin (Hoffmann LaRoche, Grenzach-Whylen, Germany) was stimulated with 10 /Lg/mLLPS from Salmonella abortus equi (Sigma-Aldrich, Deisenhofen, Germany), After24 hours at 37°Cvials were shakenand centrifuged (300 g,5 minutes).
Cytokines were measured in serum or supematants by ELISA based on antibody pairs against lL-lra, lL-2, G-CSF (R&D, Wiesbaden, Germany), lL~4 (Pharmingen, Hamburg, Ger- many), TNF-a, IFN-y (Endogen,Biozol, Eching,Germany), and sTNF-RI and 2 (Bender MedSystems,Vienna,Austria). Cvreactiveprotein was measured with the immunoturbidimet- ric kit Tina-quant CRP (Roche Diagnostics, Germany).
Peripheral Blood Mononuclear Cell (PBMC) Preparation
PBMCs were prepared from 25 mL heparinized (10 IU/mL) whole blood drawn from the cubital vein or central venous catheters at 8 AM(±1 hour) into a syringe (B. Braun Medical AG, Emmenbruecke, Switzerland)using a standard Ficoll-Hypaque (Biochrome, Berlin, Germany) densitygra- dient. The blood was layered on top of the Ficoll Hypaque at a 2: 1 ratio in 50-mL tubes and centrifuged for 20 minutesat 500 g at room temperature. PBMCs were collected from the interface, washed twice in HBSS (Gibco, Eggenstein, Ger- many), and resuspended in either RPMI 1640 (Gibco) con- taining 10% fetal calf serum (Vitromex, Vilshofen,Germany) and 0.1mg/mLgentamycin (Merk,Dannstadt,Germany) or phosphate-buffered saline (B. BraunMedical AG) containing 0.1 % sodium azide. Viability of PBMC was consistently
>95% as determined by trypan blue exclusion.
PBMC Culture
Cells (2 X 106 cells/mL) were stimulated with either LPS (10 /Lg/mL)or phytohemagglutinin (PHA HA161l7; 2,5 p.g/mL; Wellcome, Burgwedel, Germany)and incubated for 24hours or 48 hours (37°C, 6% CO2, and 90% humidity), Cell-free supernatants were collected and storedat - 80°C until assayed for cytokine production.Lymphocyte prolifer- ation over 48 hours in response to PHA was determined by 3H-thymidine incorporation as described.i"
Surface Receptor Expression
PBMCs resuspended in phosphate-buffered saline/
0.1 % sodium azide were double labeled with 1p.g/106cells of anti-CD 14-fluorescein-isothiocyanate (FITC) (RM052;
mouse IgG2a) and anti-HLA-DR-phycoerythrin(PE) (Immu- 357;mouse IgGl) or with corresponding control antibodies, all from Immunotech (Marseille, France). Samples werein- cubated for 30 minutes on ice in the dark, and then washed twice with phosphate-buffered saline/G. 1% sodium azide;
Samples were kept on ice in the dark and all measurements were analyzed within 30 minutes after completing the stain- ing procedure with a Coulter EpicsXL (Beckman Coulter
GmbH, Krefeld,Germany) fitted with a 488-nm argon laser and filter settings for FITC (530 nm wide bandpass filter) and PE (575 mn dichronic filter). After appropriate instrument settings and spectral compensations were performed, the instrument settings were not changed and stability was reg- ularly checked. A minimum of 10,000 events was assessed using log-amplified fluorescencesignalsand linearly ampli- fied side- and forward-scatter signals. XL2 software (Beck- man Coulter) was used to analyze the data.
Statistics
Statistical analysiswas performed by one-way analysis of variance followed by Tukey test or Dunn's test with SigmaStat program. P values less than 0.05 were considered significant (*P :5 0.05. >l<*P ::50.001).
RESULTS
Filgrastim treatment was welltolerated in all patients.
No significant differences were observed between the two Filgrastim treatment regimens; therefore, the groups were combined in the analyses presented.
As expected, Filgrastim initiated a rapid neutrophilia, whichelevated the total leukocyte counts to 2 to 3 times placebo values throughout the treatment period (Fig. 2a). Monocyte counts were slightly increased in the treatment group over preoperative values, while these countsremained stable in placebo blood. Interestingly, the slump in lympho- cyte counts observed after the operation ill placebo-treated patients (50% and 57% of preoperative values on days
+
Iand
+
3, respectively) was less dramatic and more short-lived in Filgrastim-treated patients(67% and 115% ofpreoperative counts on days+
1 and+
3). Furthermore, Filgrastim treat- ment induced increased serum levels of lL-l ra and soluble TNF-receptors p55 and p75, while placebo levels were not affected by the surgical interventions (Table 2).The postoperative acute phase reaction indicated by C-reactive protein and G-CSF levels in theserumwas blunted in Filgrastim-treated patients (Fig. Zb,c), Filgrastim treatment further decreased the severity and duration of monocyte inactivation: the attenuated decrease in HLA-DR expression on monocytes from Filgrastim-treated patients (Fig. 3a) cor- related with a better maintained capacity for release of TNF-a,lL-6,and IL-lra in response to LPS stimulation ex vivo in the Filgrastim treatment group (Fig.3b-d).
PHA-induced Iymphocyte proliferation dropped to half of preoperative values I day after surgery in the placebo group butwas increased in the Filgrastim-treatedgroup over preoperative values (Table 3). Also, the release of the THI Iymphokine lL-2 and IFN-y, which also dropped ill the placebo group after surgery. was increased in the Pilgrastim- treated group over placebo values. Furthermore, the lL-2/IL-4 ratio increased in treated patients compared with the placebo group after surgery (Table 3).
30
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.* *.
.*
**
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TABLE 2. Filgrastim-Treated Patients Have Higher Circulating Anti-inflammatory Mediators After Surgery
Patients received placebo or Filgrastim perioperatively, The quotients of day+I serum levels afIL-IRA, sTNF-RI, and RI! over preopcrative (day -2) levels arc given in percent ± SEM;*,"P:$ 0.05;ns, not significant, relative to placebo values.
Filgrastim
195± 24*
355± 54(ns) 182± 17*
Placebo
135 ± 15 240± 24 136± ID IL-lra(%)
sTNF-RI (%) sTNF-R2 (%)
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A list of adverse events in both treatment arms is documented (Table 4). As shown, 6 of 20 patients in the control group had infectious complications (30%), including 2 lethal outcomes, compared with 5 (13%) infectious com- plications that were observed in 40 Filgrastim-treated pa- tients. Although the blood loss of 1650 :t 266 mL during
surgery was more severe in the Filgrastim-treated group (1078 ± 164 mL in the placebo group, not significant), the average time Filgrastim-treated patients spent in the intensive care unit tended to be less (64 ± 26 hours) than placebo treated patients (156 ± 103, not significant). The average hospitalization time did not differ significantly between the two groups
DISCUSSION
Trauma induced by extensive surgery may initiate an exaggerated acute phase reaction coupled with monocyte deactivation and lymphocyte anergy.2,27 This combination severely compromises the immune system, predisposing the patient to infection and sepsis. When the time of surgery is known, sepsis prophylaxis could be administered. However, such prophylactic strategies should neither block the normal, protective inflammatory reaction to bacterial invasion nor preactivate the immune cells.
In this trial, rhG-CSF was administered prophylacti- cally before and after surgery and thus represents the first investigation, to our knowledge, with a studydesign like.this.
We found that rhG-CSF resulted in increased levels of natural circulating antagonists ofTNF-a and IL-l, ie, TNF-R p55/
p75 and IL-lra, in rhG-CSF treated patients, thus increasing the threshold for aninflammatory reaction to occur. G-CSF administration to healthy volunteers" also resulted in a mas- sive increase of counterregulatory and anti-inflammatory cy- tokines such as soluble TNF-receptors and IL-lra. While G-CSF administration to healthy volunteers also caused a reproducible increase of proinflammatory mediators such as TNF-a after ex vivo and in vitro stimulation with LPS, the increase of their antagonistic molecules was significantly higher, thus indicating that in general the impact of G-CSF on anti-inflammtory cytokine patterns appears to be more pro- nounced than its effectonproinflammatory cytokine patterns.
While this observation was corroborated in our study for TNF-a versus TNF receptors, conversely we could not con- firm down-regulating effects of G-CSF in general on proill- flammatory lymphokines like IL-2and IFN-')I, respectively an
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FIGURE 2. Perioperative Filgrastim treatment blunts trauma- induced acute phase reaction. Patients received placebo (empty bars) or Filgrastim (black bars) perioperatively. Total leukocyte counts (a), C-reactive protein (b) and G·CSF (c) serum concentrations are shown as means ± SEM*ps 0.05;
**ps 0.001, relative to placebo values.
TABLE 3. Perioperative Fllgrastim Treatment Prevents Lymphocyte Anergy and MaintainsTHl-TH2 Balance
Placebo Filgrastlm
Proliferation(%) 48 ± 5 165±41'"
1L-2(%) 45 ± 6 168 ± 35'"
•• ••
1FN-1'(%) 64 ± 10 295 ± 159Ratio 1L-2/1L-4 0,8 ± 0,2 2,1 ± OAt
3
-2 1 3
days relative to surgery
D .
' - -
up-regulatory effect on IL-4or IL-lO as described by other investigators.9,2 7.2 8 These contrasting (respectively) inconsis- tent findings are to be explained through the different timing (continuous vs. pretraumatic or post-traumatic) of G-CSP administration.A very striking examplefor the most differ- ential effects that are exerted through G-CSP 011 cytoklne release has been found by Pajkrt etalin humanendotoxcmia when G-CSP was administered eitherintravenously 2 hours or subcutaneously 24 hours before endotoxin." When admin- istered 2 hours before endotoxin,G-CSFactuallyaugmented an LPS-induced inflammatory cytokine response with high IL-6 levels, whereas when given 24 hours before LPS chal- lenge, G-CSF attenuated the LPS-inducedproinflammatory state and caused down-regulation of inflammation.
Inourstudy, the trauma-induced acute phase response, as represented by C-reactive protein measurements, was less in rhG-CSP-treated patients. Therefore, the inflammatory response to trauma was alleviatecl but not completely abol- ished by rhG-CSF treatment.Asimilar observationoccurred for the anti-inflammatory counterreaction: Serum levels of anti-inflammatory IL-l ra were significantly increased in rh- GCSF-treated patients ill comparison to controls, and post- operative HLA-DR expression on monocytes from l'hG-CSF- treatedpatients was 110t suppressed compared with controls.
Thus, the capacity of monocytes to release TNF-a when stimulated with endotoxin ex vivo was increased in rhO-CSP- treated patients over controls. Taken together,allmarkers for monocyte deactivation were improved by rhG-CSF treat-
Patients received placebo or Filgrastimperioperatively.PHA induced lL-2 release, IFN-yrelease and proliferation aredisplayed as quotientsof day+I values over preoperative (day -2) baseline values in percent :t SEM. T1y ru2 balance is expressedas quotient of IL-2over IL-4 (ratio);
valuesarcdemonstratedasmean ± SEM.
*p <0,001 versus placebo, tp =:0.05 versus placebo.
FIGURE 3. Perioperative Filgrastim treatment blunts trauma- induced monocyte deactivation. Patients received placebo (empty bars) or Filgrastim (black bars) perioperatively. a:
HLA-DR expression on monocytes. LPS-induced TNF-a (b), IL-6 (c), and IL-1 ra (d) releasein diluted whole blood. Data represent means ± SEM. *P:s; 0.05; **P:5 0.001, relative to placebovalues.
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TABLE4. Perioperative rhG-CSF Administration: Eventful Clinical Course
List ofsepsis/non-sepsis-related adversepostoperativecoursesin both treatmentarms.Sepsisand organ failure were definedaccordingto the recommendations of the American CollegeofChest Physicians/Society of CriticalCare.3~ Minorinfectiouscomplications were respiratoryinfection (n= 2)and cystitis (n
=
1).REFERENCES
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In non-neutropenic post-traumatic/postoperative pa- tients with ahigh risk ofsepsis,as reportedby Weiss etal,37 none of thepatients treated with rhG-CSFdeveloped sepsis;
however,threepatients in the control group did.As well in our study, although the number of patients was small,there wasa difference in outcomebetweenplaceboandrhG-CSF- treated patients. Of20 placebo patients, 4 developed sepsis withmultiorgan failureand 2ofthem died,but in 40rhG- CSF treated patients, only3 developed sepsis with organ failure and all recovered (Table4).
Overrecent years,we haveunderstood through amul- titudeof studiesthat traumanotonly elementarily alters the specific performance of each cell type, but probablymore importantly, it affectsthelossofcontrol capacityand mod- ulatory surveillance that physiologically monocyte and T cells possess for each other withina number of regulatory loops. Thisloss of regulatory functionoccurs instantaneously at themomentofinjuryand probably,1110Stimportantly,leads to irreversible monocyticdeactivation.We had to learn that reprogramming cellular dysfunctionaftertraumaticstressis not possible.Rather,we haveto supplythebiologicsystem with a sufficient quantity of functionally matureleukocytes that allow a timelynormalizationofthe immunoinflammatory performance. As this study shows, the administration of humanrecombinant growth factor,namely,G-CSF,cancen- trally accomplish the conservation of an innate immune system also under most stressful conditions such as major surgery, thus preventing life-threatening clinical scenarios such as second-hit multipleorgan failure. We do believethat further trialsof rhG-CSF in sepsis prophylaxis, perhaps in even higher risk populations,are warranted.
Control I'hG-CSF (n = 20) (n
=
40)6 8
4 3
2
2 3
1
I 2
3 I 1 Eventfulclinicalcourse
Septicmultipleorganfailure Nonsurvivors
Survivors Local abscess Minorinfection
Non sepsis-related deaths Stroke
Liver failure-status postpartial hepatectomy
Necrosis ofinterposedcolon
ment, The same was observed for markers of lymphocyte anergy:lymphocyteproliferativecapacity and TH1-TH2bal- ance,whichwerealtered in placebo patients,but maintained in rhG-CSF-treatedpatients after surgery.
G-CSF, a hematopoieticgrowth factor, has to be con- sidered as a multipotent molecule that exerts its effects not only directly on granulocytes but also on other leukocyte lineages like monocytes and T lymphocytes,as shown in this study. The glycoproteinG-CSF is presentat low concentra- tions (-25 p,g/mL) in the scrum of healthy volunteers."
Several studies have shown that G-CSF plasma levels are increased following trauma and the acute phase of infec- tion.3 1,32Therapeutic administration oflow-dose G-CSF (10 mg/kg) into healthyvolunteers,compared with thedose of 15 mg/kggivenin out' study, elevated the serum G-CSF tolevels in the upper range of those reached by endogenous produc- tion duringinfection.33 Interestingly,clinical examinations showed that elevated G-CSF levels at the onset of sepsis decreasedsignificantly.within a few days in survivors but remained persistently increased in nonsurviving sepsis pa- ticnts.33In contrast,patients who do not respond toinfection with increased G-CSF production have a worse prognosis than patientswho respond with G-CSFproduction.l"Thus,it can be believed that therapeutic administration ofG-CSFto trauma patients may be beneficialto the hostby increasing or acceleratingthe response to inflammation orinfection.
At present, exogenous G-CSF is widely used clinically and has been provento be effective andsafe inreducing the incidence of infection and sepsis in neutropenicand non- neutropenic immunocompromised patients.34-36 The safety
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