G-CSF immunotherapy for treatment of acute disseminated murinemelioidosis

Kellie Powell a, Glen Ulett b, Robert Hirst a, Robert Norton c;�

a Department of Microbiology and Immunology, James Cook University, Townsville, Qld, Australiab North Queensland Clinical School, The Townsville Hospital, Townsville, Qld, Australia

c Department of Microbiology, QHPS, Townsville Hospital, Townsville, Qld 4814, Australia

Received 26 February 2003; received in revised form 1 June 2003; accepted 2 June 2003

First published online 26 June 2003

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is an important pathogen in tropical regions of Australia and SoutheastAsia. Antibiotic therapy can be ineffective in patients with acute septicaemic melioidosis. It has been proposed that adjunctiveimmunotherapy using granulocyte colony stimulating factor (G-CSF) combined with antibiotics may provide an alternative approach toantibiotics alone. We have developed a murine model for melioidosis that allows novel treatment approaches to be investigated. Thisstudy looked at the potential for murine G-CSF therapy both alone and as an adjunct in the treatment of acute disseminatedB. pseudomallei infection in BALB/c mice. A number of therapeutic variables involving ceftazidime and recombinant murine G-CSF werestudied. Surviving mice were sacrificed and splenic bacterial loads were determined. Combining recombinant murine G-CSF withceftazidime offered no advantage over ceftazidime alone. Pre-treatment with recombinant murine G-CSF did not demonstrate asignificant benefit. This would suggest that adjunct immunotherapy using G-CSF is of limited benefit.6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Melioidosis ; Granulocyte colony stimulating factor; Murine model

1. Introduction

Burkholderia pseudomallei, the causative agent of melioi-dosis, is a saprophyte found in the soil in endemic areas.Both man and animals are thought to contract the infec-tion by inoculation, inhalation or ingestion of this envi-ronmental organism in contaminated soil or water [1,2].Regional endemic foci of this disease are largely con¢nedto Southeast Asia and tropical Australia between latitudes20‡S and 20‡N.

The pathogenesis of melioidosis remains poorly under-stood [2,3]. In terms of clinical presentation, three generalcategories of infection are recognised: acute, subacute, andchronic [2]. Acute melioidosis commonly presents as afulminant septicaemia, often resulting in death within afew days of exposure. Mortality rates for the acute septi-

caemic form of melioidosis remain high, ranging from 42%in Thailand to 72% in Singapore [2].

Current antibiotic therapy regimens for acute septicae-mic melioidosis typically involve ceftazidime in variouscombinations with chloramphenicol, co-trimoxazole, dox-ycycline and/or amoxycillin/potassium clavulanate. De-spite intensive and often prolonged therapy, mortalityand relapse rates remain high [4,5].

We have previously demonstrated that BALB/c mice arehighly susceptible to infection with virulent B. pseudomal-lei [3,6,7]. Disease in BALB/c mice involves a rapidly in-creasing bacteraemia and substantial bacterial growth inliver and spleen, resulting in host death within 96 h afterinfection. Assessment of bacterial splenic load is a param-eter that has been used to measure e⁄cacy of an interven-tional therapy [8]. BALB/c mice are therefore consideredto represent an animal model of the acute disseminated(septicaemic) form of human melioidosis and provide anappropriate in vivo model with which to assess the ther-apeutic e⁄cacy of novel chemotherapy or immunotherapystrategies.

Granulocyte colony stimulating factor (G-CSF) is a

0378-1097 / 03 / $22.00 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.doi :10.1016/S0378-1097(03)00473-7

* Corresponding author. Tel. : +61 (7) 47961111;Fax: +61 (7) 47962415.E-mail address: robert_norton@health.qld.gov.au (R. Norton).

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growth factor produced by a number of cell types, includ-ing monocytes, macrophages, ¢broblasts, stromal cells andendothelial cells. It has a number of actions includingstimulation of mitogenesis and di¡erentiation of commit-ted stem cells into mature polymorphonuclear leukocytes,mobilisation of mature cells into the circulation and en-hances chemotaxis, phagocytosis and oxidative activity [9].G-CSF exerts anti-in£ammatory e¡ects on monocytes bylowering the release of pro-in£ammatory cytokines whileincreasing the release of anti-in£ammatory mediators. Re-combinant human G-CSF has been shown to be e¡ectivein reducing the incidence of infections in patients withneutropenia [10]. The use of recombinant human G-CSFin non-neutropenic patients with sepsis has also been in-vestigated [9]. The e⁄cacy of G-CSF in a¡ecting survivalin community-acquired pneumonia and early onset sepsishas not been established. Two studies have failed to dem-onstrate improvements in survival with recombinant hu-man G-CSF. One involved 480 patients with multilobarcommunity-acquired pneumonia and the other involved 44preterm neonates with a clinical diagnosis of early onsetsepsis [11,12]. Another recent study was a prospective,non-randomised study looking at the use of recombinanthuman G-CSF in severe community-acquired septic shock.A total of 36 patients were treated with recombinant hu-man G-CSF and compared with historical controls. Hos-pital survival improved from 31% to 73%. Of the 36 pa-tients, six had respiratory failure and septic shock due toB. pseudomallei [13]. The hospital survival in this sub-group was improved dramatically from 5% to 100%. Itwas because of this discrepancy in outcomes in these pub-lished reports that this study was initiated.

The aim of this study was to investigate the e⁄cacy ofrecombinant murine G-CSF immunotherapy alone and asan adjunct to ceftazidime therapy for treatment of acutedisseminated B. pseudomallei infection in a BALB/c mousemodel.

2. Materials and methods

2.1. Bacteria and infection of mice

The strain of B. pseudomallei used in this study wasNCTC 13178. The identity of the isolate was con¢rmedby colonial morphology on Ashdown agar and API 20NE(bioMerie¤ux, La Balme, France). B. pseudomallei wasgrown in brain heart infusion broth (Oxoid, Hampshire,UK) at 37‡C for 18 h and stored in 1-ml aliquots at380‡C in 20% glycerol. When required, an aliquot wasthawed at 37‡C and subcultured onto Ashdown agar.After 48 h incubation at 37‡C, three to ¢ve colonieswere used to prepare a single cell suspension in sterilephosphate-bu¡ered saline (PBS) equivalent to 0.5 McFar-land (optical density of 0.18 at 650 nm; Multiskan EX355v1.0, Labsystems, Finland). Appropriate dilutions were

prepared in PBS to achieve a concentration of 1.5U103

colony forming units (CFU) per ml. Inbred BALB/c mice(8^16 weeks) were administered 300 CFU intravenously in200 Wl of PBS. Colony count checks were performed by astandard method after 48 h incubation at 37‡C.

2.2. Animal groups

35 BALB/c mice were divided into seven groups of ¢veand designated 1^7. Groups 1^4 were involved in treat-ment therapy programmes while groups 5 and 6 were in-volved in pre-treatment therapy programmes. The ¢nalgroup of mice, designated group 7, was inoculated intra-venously with PBS only and received subsequent intraper-itoneal injections of murine G-CSF once daily. These micerepresent an uninfected treatment control group to deter-mine any toxicological e¡ects murine G-CSF may have inBALB/c mice. Treatment therapy programmes are summa-rised in Table 1.

2.3. Treatment programmes

At 48 h post-inoculation with B. pseudomallei treatmentprogrammes for groups 1^4 began. These programmeswere designated 1^4 where programme 1 represented in-fected untreated controls that received subsequent injec-tions of PBS only, 2 was with recombinant murine G-CSF (AMGEN, CA, USA), 3 was with ceftazidime (GlaxoSmith Kline, Australia) and 4 was with recombinant mu-rine G-CSF and ceftazidime.

2.4. Pre-treatment

Mice in groups 5 and 6 were allocated to the recombi-nant murine G-CSF pre-treatment programmes. Pro-gramme 5 was recombinant murine G-CSF pre-treatmentonly, with recombinant murine G-CSF administered intra-peritoneally at 48 h and 24 h prior to inoculation withB. pseudomallei ; programme 6 was recombinant murineG-CSF pre-treatment with recombinant murine G-CSFadministered intraperitoneally at 48 h and 24 h prior toinoculation with B. pseudomallei with an additional anti-biotic therapy programme of ceftazidime 48 h post-inocu-lation.

Table 1Treatment groups of BALB/c mice (¢ve per group)

Group Pre-treatment Inoculation Treatment

Group 1 PBS only B. pseudomallei PBS onlyGroup 2 PBS only B. pseudomallei G-CSF onlyGroup 3 PBS only B. pseudomallei Ceftazidime onlyGroup 4 PBS only B. pseudomallei G-CSF and

ceftazidimeGroup 5 G-CSF 48 and 24 h B. pseudomallei PBS onlyGroup 6 G-CSF 48 and 24 h B. pseudomallei Ceftazidime onlyGroup 7 PBS only PBS only G-CSF only

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Recombinant murine G-CSF and ceftazidime were re-constituted in sterile PBS as per the manufacturer’sinstructions. Working solutions of recombinant murineG-CSF and recombinant murine G-CSF with ceftazidimewere made fresh daily. Working solutions of ceftazidimewere stored in 1-ml syringes at 380‡C until use. Recombi-nant murine G-CSF was administered intraperitoneallyonce daily at 100 Wg kg31. Ceftazidime was administeredintraperitoneally twice daily at 600 mg kg31, 10 times therecommended human dose rate for ceftazidime. The doserate for ceftazidime was calculated based on plasma levelsof antibiotics that were determined over a 12-h period byhigh performance liquid chromatography [8].

At 10 days post-inoculation with B. pseudomallei, eachof the ¢ve mice per group was euthanised and the bacterialload in spleen was determined as previously described [6].Control mice (group 1) and recombinant murine G-CSFtreatment only (group 2) were not assessed beyond day 3due to progressive illness. Recombinant murine G-CSFpre-treatment only mice (group 5) were not assessed be-yond day 4 due to progressive illness. Spleen was homo-genised in 10 ml of PBS using a Stomacher (Townson andMercer, UK). Serial dilutions were prepared in PBS andthese were dispensed onto Ashdown agar (Micro Diagnos-tics, Australia) to determine viable B. pseudomallei counts.This was expressed as CFU per ml of splenic homogenate.Clinical parameters such as temperature and weight werenot recorded, as bacterial counts in spleen provide a re-producible measure of disease severity in this model [6].

2.5. Statistical analysis

P values were calculated using a t-test (SPSS for Win-dows version 10.0.7, SPSS Inc., USA) with P values6 0.05 considered signi¢cant.

3. Results

The splenic bacterial loads from the di¡erent treatmentgroups are expressed as CFU ml31 of splenic homogenateusing a exponential scale in Fig. 1. Treatment using re-combinant murine G-CSF alone was no better than thePBS control (P=0.6104), with all mice moribund within72 h. The best outcome was seen in the group 6, pretreatedwith murine G-CSF and subsequently treated with cefta-zidime (P6 0.0001, Av CFU ml31 = 33.33), which wasonly slightly better than treatment using ceftazidime alone(group 3; P6 0.0001, Av CFU ml31 = 56.67). The com-bined treatment of recombinant murine G-CSF and cefta-zidime (group 4), 48 h post-infection reduced the splenicbacterial load but was not as e¡ective as ceftazidime treat-ment alone (group 3; P=0.0032). Pre-treatment with re-combinant murine G-CSF alone did not reduce the splenicbacterial load or mortality, as all mice died or were eu-thanised within 96 hrs. Recombinant murine G-CSF wasfound to have no adverse toxicological a¡ects on theBALB/c mice in this study.

4. Discussion

This pre-clinical animal study is consistent with previousclinical trials which show no bene¢t in the use of adjunc-tive G-CSF therapy with antibiotics in the management ofsevere sepsis [11,12]. Of interest is the ¢nding here, thatpre-treatment with recombinant murine G-CSF improvedbacterial killing (group 6 vs group 4), which is in concord-ance with a study which evaluated the place of G-CSF inexperimental peritoneal sepsis [14]. It is acknowledged thatthese two groups are not directly comparable. Recombi-nant murine G-CSF enhanced the e⁄cacy of ceftazidime(P=0.0032) when administered prior to B. pseudomalleiinfection in this model (group 6 vs group 4), by reducingbacterial numbers in the splenic homogenate compared totreatment with ceftazidime alone, and may be related tothe recruitment of polymorphonuclear leukocytes prior tothe onset of sepsis, resulting in enhanced killing.

Therapy treatment programme 6 (pre-treatment withrecombinant murine G-CSF and post-treatment with cef-tazidime) provided the most signi¢cant reduction in thesplenic bacterial load and prevention of mortality. Therewas, however, no signi¢cant di¡erence between the splenicbacterial loads in mice treated in therapy programme 6and those mice treated in therapy programme 3 usingceftazidime only (P=0.4935). It can be concluded thattreatment with ceftazidime only will provide the same de-gree of bacterial clearance as those mice pre-treated withrecombinant murine G-CSF (group 3 vs group 6), withoutthe additional impracticality of a pre-treatment regime.While the measurement of splenic bacterial load may notallow direct comparison with other ‘clinical’ measures inpatients such as weight and temperature, it does provide

0

1

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3

4

5

6

7

8

1 2 3 4 5 6

Treatment Program

Sp

len

ic L

oad

(lo

g C

FU

/ml)

10

Fig. 1. A comparison of chemotherapeutic and immunotherapeutictreatment regimens involving ceftazidime and G-CSF for the treatmentof acute disseminated B. pseudomallei infection in BALB/c mice. Bacteri-al loads in spleen (log 10 CFU ml31 ; n=5) of mice in the various treat-ment groups were determined 7 days following challenge with B. pseudo-mallei, as a measurement of disease severity.

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important information as to the clinical state of mice inthe model used the current experiment [6]. The BALB/cmouse represents a unique model for experiments designedto test novel therapeutic strategies in melioidosis. Possiblefuture uses of the model would be a comparison of therelative e⁄cacy of ceftazidime and meropenem in thetreatment of this condition.

As such, the results from this study only provide pre-liminary data and useful information on which to basecontrolled human clinical trials.

Acknowledgements

We gratefully acknowledge the support of AMGEN(USA) in providing murine G-CSF.

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[2] Currie, B.J. (2000) Melioidosis : An Australian Perspective of anEmerging Infectious Disease. Recent Advances in Microbiology,Vol. 8, pp. 1^75. The Australian Society of Microbiology, Mel-bourne.

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