Effect of adjunctive treatment with gamma interferon against Pseudomonas aeruginosa pneumonia in neutropenic and non-neutropenic hosts

International Journal of Antimicrobial Agents 24 (2004) 21–27

Effect of adjunctive treatment with gamma interferon against Pseudomonas aeruginosa pneumonia in neutropenic and non-neutropenic hosts Chinedum P. Babalola a , Charles H. Nightingale a,b , David P. Nicolau a,c,∗ a

Center for Anti-Infective Research and Development, Hartford Hospital, 80 Seymour Street, Hartford, CT 06102-5037, USA b Office for Research, Hartford Hospital, Hartford, CT 06102, USA c Division of Infectious Diseases Hartford Hospital, Hartford, CT 06102, USA Received 29 October 2003; accepted 19 March 2004

Abstract To evaluate the adjunctive effect of interferon-gamma (IFN-␥) in treatment of Pseudomonas aeruginosa pneumonia, neutropenic and non-neutropenic mice received LD100 of the organism intratracheally, followed by subcutaneous administration of IFN-␥, ceftazidime (TAZ) or a combination of both agents 2 h post-inoculation. Treatment with IFN-␥ alone showed no significant increase in survival when compared with control. Addition of IFN-␥ to TAZ resulted in no significant change in survival compared with TAZ alone. Survival in TAZ and TAZ + IFN-␥ groups, was significantly higher than in control and IFN-␥ groups. This suggests that adjunctive treatment with IFN-␥ in combination with ceftazidime may not be more beneficial than the antibiotic alone when managing acute P. aeruginosa pneumonia in both immunocompromised and immunocompetent hosts. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Interferon-gamma; Pseudomonas aeruginosa; Ceftazidime; Immunomodulators

1. Introduction Nosocomial pneumonia has the highest crude mortality rate of all hospital-acquired infections [1,2]. Pseudomonas aeruginosa (PSA) is one of the most common causes and most virulent pulmonary pathogen associated with nosocomial pneumonia especially in the immunocompromised individuals [3,4]. PSA-induced pneumonia causes morbidity and mortality in the range of 18–61% [5,6]. When PSA is implicated, monotherapy, even with broad-spectrum antibiotics is associated with rapid evolution of resistance and a high rate of clinical failure [2]. Therefore empirical treatment for serious infections caused by PSA usually involves combination therapy, and novel strategies employing cytokine treatment may have promise. Adjunctive immunomodulatory therapies in the management of infections are a growing area of interest because potentiation of the non-specific immune system offers an opportunity to maximise resolution of serious infections [7]. ∗

Corresponding author. Tel.: +1 860 5453941; fax: +1 860 5453992. E-mail address: [email protected] (D.P. Nicolau).

Studies on an immunotherapeutic approach involving the use of interferon-gamma (IFN-␥) against gentamicin- and vancomycin-resistant Enterococcus faecalis (GVRE) have been investigated in our laboratory [7–9]. In these studies, IFN-␥ significantly augmented the antienterococcal activities of gentamicin and vancomycin in vitro as well as in vivo in the non-neutropenic (NN) mouse model. However, in neutropenic (NT) mice, the activities of the antibiotics were not enhanced by IFN-␥ combinations. IFN-␥ is reported to be the most broadly acting cytokine identified in experimental models in infectious diseases [10–12]. It has been identified as an important immunomodulator in early host defence against a variety of infections [13]. IFN-␥ acts by activating monocytes and neutrophils with resultant enhancement of their microbicidal activities through phagocytosis, although it may also modulate in vivo antibacterial activity through the induction of secondary mediators [11,12]. As a result of the promising adjunctive IFN-␥ treatment, we hypothesised that IFN-␥ could be a beneficial adjunct in the treatment of PSA pneumonia infections. Although there are several reports on merits and demerits of both endogenous IFN-␥ and administered IFN-␥

0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2004.03.019

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in infection models [14–19], administration of IFN-␥ and several other immunomodulators in combination with antimicrobial therapy has undergone limited study [7–9] and no data are available concerning combined IFN-␥ and antibiotic in the treatment of acute PSA pneumonia. Thus, the purpose of this study was to evaluate the effects of administration of IFN-␥ in combination with ceftazidime (TAZ) in the treatment of a mouse model of pneumonia induced by PSA in both neutropenic (NT) and non-neutropenic (NN) hosts after onset of infection.

Recombinant human gamma interferon (1 × 7 U/mg protein) was obtained in lyophilised form (Calbiochem, Lajolla, CA, USA) and was stored at −20 ◦ C until use. Stock solutions (10 ␮g/ml) were prepared in normal saline and stored at −80 ◦ C until use. Desired concentrations were made in normal saline from the stock solutions according to the dose required and administered subcutaneouly (s.c.) to the mice in 0.2 ml volumes. 2.4. Induction of lung infection

Female Swiss Webster mice weighing 20–25 g (Harlan Sprague Dawley, Indianapolis, IN, USA) were used. They were acclimatised for 1 week before the study, in a room with controlled temperature and a 12 h light–12 h dark cycle. The animals were allowed access to food and water ad libitum. The hospital’s Institutional Animal Care and Use Committee approved all studies and all studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.

A group of mice were rendered neutropenic (NT) by i.p. administration of cyclophosphamide 150 mg/kg on days 4 and 1 before bacterial inoculation or any further treatment [20]. The non-neutropenic (NN) group did not receive cyclophosphamide. On day 0, a suspension of PSA (PSA 27853) was prepared as described above. Lung infection was induced as described by Kim et al. [20]. Briefly, isofluorane (2%, v/v) in 100% oxygen carrier was used to induce a semi-anaesthetised state. Intratracheal inoculation was implemented to induce pneumonia by instilling a 0.05-ml bacterial suspension into the mouth and completely blocking the nares of the animal, Thus, resulting in bacterial inhalation through the mouth to the lungs. The mice were placed in an oxygen-enriched chamber for full recovery and thereafter randomised into control and various treatment groups.

2.2. Bacteria

2.5. Minimum lethal dose (MLD)

2. Materials and methods 2.1. Animals

A NCCLS quality control isolate of PSA (PSA 27853) with a ceftazidime MIC of 2 mg/l was used. The isolate was maintained in skim milk medium (Becton Dickinson, Cockeyville, MD, USA) at −80 ◦ C and sub-cultured twice onto trypticase soy agar with 5% sheep blood (Becton Dickinson) at 37 ◦ C before use in all experiments. A suspension of the PSA 27853 was prepared from the second subculture that had been incubated for between 18–24 h. and was adjusted to >4.0 Mc Farland turbidity standard in a 5% dextrose saline solution, approximating 109 CFU/ml. From this suspension, necessary dilutions were made for inoculation of the mice. The bacterial density of each run was confirmed by serial dilution and culture of an aliquot from each inoculum suspension for quality control. 2.3. Drugs and cytokine Ceftazidime (TAZ) in one-gram vials (Glaxo SmithKline, Research Triangle, Park NC, USA) were reconstituted according to the manufacturer’s instructions in normal saline. Further dilutions were made in normal saline to obtain the required dosages and administered subcutaneously (s.c.) in 0.2 ml volumes. Cyclophosphamide (Cytoxan; Bristol-Myers Squibb, Princeton, NJ, USA) was reconstituted in distilled water to obtain 150 mg/kg of body weight. The drug was dosed intraperitoneally (i.p.) in 0.2 ml volumes.

The minimum lethal dose (MLD) was determined by injecting groups of 20 mice (NT and NN) with serial dilutions of the bacterial suspension prepared above. Animals were observed and the lowest dilution of inoculum that produced 100% mortality within 3 days after infection was recorded and taken as the MLD. 2.6. Antibiotic protective dose Studies were performed to determine the dose of ceftazidme (TAZ) that reproducibly resulted in not more than 50% protection against death after infection of the mice. NT mice were inoculated with 0.05 ml of ∼ 5 × 107 CFU/ml of the organism (MLD for NT mice), while NN mice received ∼ 5 × 109 CFU/ml (MLD for NN mice). Two hours after infection, the animals were given TAZ s.c. at a dose range of 125–2000 mg/kg. A second dose of the drug was given 4 h thereafter (i.e. 6 h post-infection) making a total of two doses of TAZ. Twenty mice were used for each drug dose. Survival of animals was recorded at four time points daily for 4 days. Preliminary studies showed that by one-hour post-inoculation, the mice were fully infected. 2.7. Therapy with IFN-γ To evaluate the antibacterial activity of IFN-␥, NT and NN mice were inoculated with a MLD of the organism and randomised into treatment groups (10 mice/group) that

C.P. Babalola et al. / International Journal of Antimicrobial Agents 24 (2004) 21–27

received 0, 0.05, 0.15 or 0.5 ␮g/mouse/day for 3 days starting 2 h after infection. Control mice received normal saline. Survival was monitored daily for 5 days and percent survival as a measure of efficacy was recorded. 2.8. Combination therapy with antibiotic and IFN-γ To evaluate the effect of combination therapy, mice infected with MLD (LD100 ) of the organism were randomised into treatment groups that were given s.c. TAZ alone (2000 mg/kg × 2 doses); IFN-␥ alone (0.5 ␮g/mouse/day for 3 days), or IFN-␥ + TAZ. The control group received normal saline. All doses were initiated 2 h after infection and given in 0.2 ml volumes. 22–50 mice were used in each treatment group and animals were monitored daily for survival over 5 days. The dosage schedules used in the treatment of infected immunocompetent mice (NN) were also used for the NT mice. All combination experiments were carried out in triplicate and pooled data were used for statistical analysis. 2.9. Statistical analysis The minimum number of animals required in each treatment group to provide sufficient statistical power was determined by sample size estimation based on log rank test for a fixed time and constant hazard ratio. Kaplan Meier Plots T estimated time to death and percent survival in both NT and NN mice and the estimates of mortality were compared among treatment groups by using the log rank test. For all tests, a P value of < 0.05 was considered significant.

3. Results 3.1. Therapeutic effect of IFN-γ The neutropenic (NT) and non-neutropenic (NN) mice inoculated with PSA were injected with different doses of

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IFN-␥ alone and the survival profile obtained is shown in Table 1. Administration of various dosages of IFN-␥ produced no significant change in survival compared with control mice that received normal saline in both NT and NN groups. The rate of mortality was higher in the NN group than in the NT even though by 24 h all the control mice in NN group had died. There was no dose-related effect on survival observed with the cytokine. In order to test the safety of the IFN-␥ doses used, uninfected NT and NN mice were given the same doses of IFN-␥ previously used in the infected hosts (0–0.5 ␮g/mouse/day × 3 days), however, none of the mice in both groups died, even with the highest dose of 0.5 ␮g/mouse. Therefore the highest dose of 0.5 ␮g/mouse of IFN-␥ was chosen for the combination therapy trials. 3.2. Combination therapy with IFN-γ and ceftazidime To evaluate the impact of IFN-␥ on efficacy of ceftazidime (TAZ), 2000 mg/kg TAZ (×2 doses) that was associated with optimal survival and 0.5 ␮g/mouse/day × 3 days of IFN-␥ were chosen for the treatment of infected NT and NN mice. The survival rates resulting from combined administration of IFN-␥ and TAZ against PSA infection in both immunocompromised (NT) and immunocompetent (NN) hosts are shown in Table 2. In the NT mice, treatment with IFN-␥ did not significantly increase survival of infected mice with the survival percent ranging from 0% (control) to 4% (IFN-␥ alone) (P = 0.163), which is consistent with the dose range result shown in Table 1. From 3 days post-infection, treatment with TAZ alone or TAZ + IFN-␥ resulted in significantly higher survival than the control and IFN-␥ alone groups (P < 0.0001). Survival was nearly constant from 3 days post-infection to 5 days post-infection. However, at the end of 5 days, addition of IFN-␥ to TAZ produced no significant change (P = 0.91) in survival (59%) compared with treatment with TAZ alone (57%). The trend of results obtained in NT-infected mice was also obtained in NN-infected mice (Table 2). While the survival in control and IFN-␥ alone were comparable (P = 0.31)

Table 1 Survival of neutropenic and non-neutropenic mice after infection with 100% lethal dose (LD100 ) of Pseudomonas aeruginosa (PSA 27853) and treatment with doses of interferon-␥ Host

IFN-␥ dose (␮g/mouse)a

n

Neutropenic

0b 0.05 0.15 0.5

10 10 10 10

0b 0.05 0.15 0.5

10 10 10 10

Non-neutropenic

a b

Number of mice (%) survival 12 h

24 h

48 h

72 h

96 h

120 h

10 (100) 10 (100) 10(100) 10(100)

7 9 10 9

(70) (90) (100) (90)

1 2 2 2

(10) (20) (20) (20)

1 0 1 1

(10) (0) (10) (10)

0 0 1 0

(0) (0) (10) (0)

0 0 1 0

(0) (0) (10) (0)

0 1 0 1

(0) (10) (0) (10)

0 1 0 1

(0) (10) (0) (10)

0 1 0 1

(0) (10) (0) (10)

0 1 0 1

(0) (10) (0) (10)

0 1 0 1

(0) (10) (0) (10)

6 7 6 7

(60) (70) (60) (70)

Doses of IFN− were administered daily for 3 days after infection. Control mice received normal saline alone daily for 3 days post-infection.

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Table 2 Survival of neutropenic and non-neutropenic mice after infection with 100% lethal dose (LD100 ) of Pseudomonas aeruginosa (PSA 27853) and treatment with ceftazidime alone or in combination with IFN-␥ Host

Treatment

Neutropenic

Controla

Non-neutropenic

a b c d

n

Number (%) of mice surviving after infection Day 1

Day 2

Day 3

Day 4

Day 5

IFN-␥b TAZc TAZ + IFN-␥d

22 24 49 49

13 18 49 49

(59) (75) (100) (100)

3 5 44 42

(14) (21) (90) (86)

1 1 29 32

(5) (4) (59) (65)

0 1 28 30

(0) (4) (57) (61)

0 1 28 29

(0) (4) (57) (59)

Controla IFN-␥b TAZc TAZ + IFN-␥d

22 22 50 50

6 7 32 37

(27) (32) (64) (74)

2 4 21 30

(9) (22) (42) (60)

1 2 13 18

(5) (9) (26) (36)

1 2 12 18

(5) (9) (24) (36)

1 2 12 18

(5) (9) (24) (36)

Mice received normal saline. Mice received 0.5 ␮g/mouse IFN-␥ daily for 3 days starting 2 h after infection. TAZ (ceftazidime) was administered as 2000 mg/kg × 2 doses. In combination therapy, TAZ and IFN-␥ were administered at the same doses as in monotherapy.

addition of IFN-␥ to TAZ resulted in an increase in survival (36%) compared to treatment with TAZ alone (24%) but the 12% difference observed was not statistically significant (P = 0.076). To confirm the results obtained, Kaplan Meier Plots were performed on the data obtained as shown in Fig. 1 for NT group and Fig. 2 for NN group of mice. In the NT groups

but not in NN, survival curves of TAZ and IFN-␥ + TAZ are nearly superimpossable, while in both NT and NN groups, survival curves of control and IFN-␥ alone groups are nearly superimpossable. In general, survival % was more in the neutropenic mice than in the non-neutropenic mice after drug alone and drug + IFN-␥ combination therapy (Table 2).

Fig. 1. The efficacies of treatment with ceftazidime and IFN-␥ separately or as a combination of both agents on the survival profiles of neutropenic mice infected with 100% lethal dose of Pseudomonas aeruginosa pneumonia.

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Fig. 2. The survival profiles of non-neutropenic mice following infection with Pseudomonas aeruginosa pneumonia and treatment with ceftazidime and IFN-␥ separately or in combination.

4. Discussion The alarming increase in antimicrobial resistance noted in nosocomial settings in recent years and being facilitated by extensive use of different classes of antibiotic provides a compelling argument in favour of combination therapy with antibiotic and cytokines. Interferon is the principal cytokine for activating human macrophages and enhances phagocytosis [21]. The major findings in this study were that interferon gamma had no benefit on its own and that a combination of IFN-␥ as an adjunct to ceftazidime resulted in no improvement in therapy of PSA pneumonia in both immunocompromised and immunocompetent hosts after onset of infection. A few studies on have shown beneficial effect of IFN-␥ in enhancing antibiotic therapy in animal infection models [7,9,22]. The study by Onyeji et al. [9] showed adjunctive efficacy of IFN-␥ when combined with gentamicin and vancomycin in the treatment of GVRE in non-neutropenic (NN) mice but not in neutropenic mice. The lack of effect of IFN-␥ alone and in combination with TAZ in the present study may be attributable to several factors. Previous studies in our laboratory and elsewhere have shown that cytokine effect against bacterial infections is influenced by the level of infectious burden [7,9,23–25]. The MLD (LD100 ) of PSA used in the present study could be overwhelming for the hosts coupled with the virulence characteristics of PSA in acute infection [4]. In the study by Onyeji et al. [9] mice inoculated with LD70 of the organism were much more protected by IFN-␥ (71% survival)

than those infected with LD100 (21% survival). Slight differences in inoculum size (within the same log CFU/ml) were reported to affect survival outcome in a multidrug resistant enterococci (MDRE) infection model [7]. This infection burden coupled with the bacterial virulence might have knocked out the effect of dose ranging of IFN-␥ in the present study. Dose-related effect has been reported with IFN-␥ in some other bacterial infections that have employed lower inoculum sizes of bacteria [7,9,15]. While Hershman et al. [15] obtained dose effect up to 7500 U/day of IFN-␥ (equivalent to 0.75 ␮g/day) Onyeji et al. [9] obtained an optimum IFN-␥ dose of 0.15 ␮g/day. The toxicity test carried out with the IFN-␥ doses proves the safety of the cytokine in this model. The doses used were also comparable with the IFN-␥ doses of 50–300 ␮g/m2 used in humans [11]. Unexpectedly, a seemingly poorer survival outcome was observed in the NN mice compared with neutropenic probably due to the inoculum effect (Tables 1 and 2). Although both groups received MLD of the PSA, the MLD of PSA in NN was 109 CFU/ml while that in NT was 107 CFU/ml. The lack of cytokine effect in this model may also be connected to the time of initiation of therapy after infection. Unlike in most studies where beneficial outcome was reported after pretreatment (prophylaxis) with IFN [14–16,26] or after concurrent administration with bacterial inoculum [7,9], IFN-␥ treatment in the present study was initiated 2 h after lung inoculation with the bacteria. This was done in order to mimic clinical situations, since patients do not present with symptoms of infection until a while after getting in contact

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with the bacteria. Pretreatment coupled with therapeutic administration of IFN-␥ was found beneficial in PSA infection in mice [16]. Onyeji et al. [9] observed beneficial outcome in GVRE model only when the IFN-␥ and/or antibiotics were administered concurrently with the bacteria, but when treatment was started 2 h after the infection, no survival was recorded in any of the treatment groups. Recent findings in patients point to the fact that delay in starting effective antimicrobial therapy for severe PSA infections tends to higher mortality [6]. The type of bacteria and infection model employed in this study might also contribute to the observed outcome. Species and strain differences with regards to the role of IFN-␥ in infectious models have been reported [14,16]. Several studies have shown the protective role of IFN-␥ in defense against Klebsiella pneumoniae infections [14,15,27] unlike PSA infections. In a comparative study, mice treated with IFN-␥ survived significantly longer than the control when the bacterial challenge was KPN, but no difference in survival was observed when the bacteria was PSA [14]. Protective effect of IFN-␥ against PSA pneumonia infection was observed in chronic infection models mimicking cystic fibrosis patients, but no protection was observed during acute infection [28]. Although the levels of cytokines in the blood or organs were not determined in this study, earlier reports have shown that excessive production of both proinflammatory cytokines (such as IFN-␥ and alpha tumour necrosis factor-␣ [TNF-␣]) and antiinflammatory cytokines (such as interleukin-10 [IL-10]) due to acute and severe infection stimuli, play a role in the pathogenesis of bacterial infection, resulting in poor clinical outcome [29–31]. During PSA infections increased levels of IL-10 is synonymous to increased IFN-␥ [19,31–33] causing exacerbation of lung injury and increase in mortality. A recent study in our laboratory showed [34] that the presence of white blood cells brought about a decrease in bacterial densities in the lungs of CBA/J mice infected with pneumococcal pneumonia, the cells excessively augmented the immune response, causing the lungs injury and death. It is not unlikely that the acute PSA infection model involved in this study might have caused imbalance between the proand counter-inflammatory response consequently causing severity of the disease and poor final outcome. The better survival profiles observed in neutropenic mice compared to non-neutropenic (Tables 1 and 2) might be partly connected to possible exaggerated levels of cytokines in the NN hosts. The study by Tessier et al. [35] showed that survival of immunocompetent CBA/J mice, which were enriched with white blood cells, was unexpectedly lower than that of immunocompromised ICR mice during pneumococcal pneumonia infection. Some other studies showed that endogenous IFN-␥ such as is present in the immunocompetent host impairs rather than augments host defence during severe pneumonia caused by PSA [17] as well as Streptococcus pneumoniae [18] unlike in the immunocompromised counterpart.

In conclusion, the present study shows that IFN-␥ provides no protection against acute P. aeruginosa pneumonia infection in both immunocompromised and immunocompetent hosts after challenge and onset of infection with MLD of the bacteria. The study also demonstrates that the efficacy of ceftazidime against P. aeruginosa pneumonia is not significantly modified by concurrent therapy with IFN-␥. The improved survival outcome obtained in the drug-treated and drug + IFN-␥-treated groups appears due to be the effect of the antimicrobial alone. Therefore, immunochemotherapy using adjunctive gamma interferon with ceftazidime may not provide a therapeutic option for the management of acute P. aeruginosa pneumonia infections in both immunocompromised and immnunocompetent hosts.

Acknowledgements This study was supported by a grant from Hartford Hospital (Grant # 128144).

References [1] Ramirez JA. The choice of empirical therapy for nosocomial pneumonia. J Chemother Suppl 1994;2:47–50. [2] Lynch JP. 3rd Hospital-acquired pneumonia: risk factors, microbiology, and treatment. Chest 2001;119:3735–845. [3] Kuikka A, Valtonen VV. Factors associated with improved outcome of Pseudomonas aeruginosa bacteremia in a Finnish university hospital. Eur J Clin Microbiol Infect Dis 1998;17:701–8. [4] Cunha BA. Nosocomial pneumonia: diagnostics and therapeutic considerations. Med Clin North Am 2001;85:79–114. [5] Maschmeyer , Braveny I. Review of the incidence and prognosis of Pseudomonas aeruginosa infections in cancer patients in the 1990s. Eur J Clin Microbiol Infect Dis 2000;19:915–25. [6] Kang C, Kim S, Kim H, et al. Pseudomonas aeruginosa bacteremia: Risk factors for mortality and influence of delayed receipt of effective antimicrobial therapy on clinical outcome. Clin Infect Dis 2003;37:745–51. [7] Nicolau DP, Onyeji CO, Banevicius MA, Li J, Nightingale CH. Effects of adjunctive treatment with combined cytokines in a neutropenic mouse model of multidrug-resistant Enterococcus faecalis septicemia. Pharmacotherapy 2001;21:275–80. [8] Nicolau DP, Onyeji CO, Banevicius MA, Li J, Nightingale CH. Effects of adjunctive treatment with combined cytokines in a neutropenic mouse model of multidrug-resistant Enterococcus faecalis septicemia. Pharmacotherapy 2001;21:275–80. [9] Onyeji CO, Nicolau DP, Nightingale CH. Bow l. Interferon-gamma effects on activities of gentamicin and vancomycin against Enterococcus faecalis resistant to the antibiotics: an in vitro study with human neutrophils. Int J Antimicrob Agents 1999;11:31–7. [10] Murray HW. Interferon-gamma, the activated macrophage, and the host defense against microbial challenge. Ann Intern Med 1988;108:595–608. [11] Murray HW. Interferon-gamma and host antimicrobial defense: current and future clinical applications. Am J Med 1994;97:459–67. [12] Roiledes E, Pizzo PA. Modulation of host defenses by cytokines: evolving adjuncts in prevention and treatment of serious infections in immunocompromised hosts. Clin Infect Dis 1992;15:508–24. [13] Farrar M, Schreiber RD. The molecular cell biology of interferon-␥ and its receptor. Ann Rev Immunol 1993;11:571–611.

C.P. Babalola et al. / International Journal of Antimicrobial Agents 24 (2004) 21–27 [14] Hershman MJ, Sonnenfeld G, Logan WA, Pietsch JD, Wellhausen SR, Polk Jr HC. Effect of interferon-gamma treatment on the course of a burn wound infection. J Interferon Res 1988;8:367–73. [15] Hershman MJ, Polk Jr HC, Pietsch JD, Kuftinec D, Sonnenfeld G. Modulation of Klebsiella pneumoniae infection of mice by interferon-gamma. Clin Exp Immunol 1988;72:406–9. [16] Pierangeli SS, Polk Jr HC, Parmely MJ, Sonnenfeld G. Murine interferon-gamma enhances resistance to infection with strains of Pseudomonas aeruginosa in mice. Cytokine 1993;5:230–4. [17] Schultz MJ, Rijneveld AW, Speelman P, van Deventer SJ, van der Poll T. Endogenous interferon-gamma impairs bacterial clearance from lungs during Pseudomonas aeruginosa pneumonia. Eur Cytokine Netw 2001;12:39–44. [18] Rijneveld AW, Lauw FN, Schultz MJ, et al. The role of interferon-gamma in murine pneumococcal pneumonia. J Infect Dis 2002;185:91–7. [19] Moore TA, Perry ML, Getsoian AG, Newstead MW, Standiford TJ. Divergent role of gamma interferon in a murine model of pulmonary versus systemic Klebsiella pneumoniae infection. Infect Immun 2002;70:6310–8. [20] Moore TA, Perry ML, Getsoian AG, Newstead MW, Standiford TJ. Divergent role of gamma interferon in a murine model of pulmonary versus systemic Klebsiella pneumoniae infection. Infect Immun 2002;70:6310–8. [21] Kim M, Zhou W, Tessier PR, et al. Bactericidal effect and pharmacodynamics of cethromycin (ABT)-773) in a murine pneumococcal model. Antimicrob Agents Chemother 2002;46:3185–92. [22] Bellardelli F. Role of interferons and other cytokines in the regulation of the immune response. APMIS 1995;103:161–79. [23] Shear HL, Valludares G, Narachi MA. Enhanced treatment of Pneumocystis carinii pneumonia in rats with interferon-gamma and reduced doses of trimethoprim-sulfamethoxazole. J Acquired Immune Defic Syndr 1990;3:943–8. [24] Onyeji CO, Nicolau DP, Nightingale CH, Bow L. Modulation of efficacies and pharmacokinetics of granulocyte colony-stimulating factor in neutropenic mice with multidrug-resistant resistant Enterococcus faecalis infection. J Antimicrob Chemother 2000;46:429– 36.

27

[25] Dallaire F, Ouillet N, Simard M. Efficacy of recombinant granulocyte colony-stimulating factor in a murine model of pneumococcal pneumonia: Effects of lung inflammation and timing of treatment. J Infect Dis 2001;183:70–7. [26] Kullberg BJ, van’t Wout JW, Hoogstraten C, van Furth R. Recombinant interferon-gamma enhances resistance to acute disseminated Candida albicans infection in mice. J Infect Dis 1993;168:436–43. [27] Pierangeli SS, Sonnenfeld G. Treatment of murine macrophages with murine interferon-gamma and tumour necrosis factor-alpha enhances uptake and intracellular killing of Pseudomonas aeruginosa. Clin Exp Immunol 1993;93:165–71. [28] Yoshida K, Matsumoto T, Tateda K, et al. Protection against pulmonary infection with Klebsiella pneumoniae in mice by interferongamma through activation of phagocytic cells and stimulation of production of other cytokines. J Med Microbiol 2001;50:959–64. [29] Johansen HK, Hougen HP, Rygaard J, Hoiby N. Interferon-gamma (IFN-␥) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats. Clin Exp Immunol 1996;103:212–8. [30] Salgado A, Boveda JL, Monasterio J, Segura RM, Mourelle M, Gomez-Jimenez J, et al. Inflammatory mediators and their influence on haemostasis. Haemostasis 1994;24:132–8. [31] Yamaguchi T, Hirakata Y, Izumikawa K, et al. Prolonged survival of mice with Pseudomonas aeruginosa-induced sepsis by rIL-12 modulation of IL-10 and interferon-␥. J Med Microbiol 2000;49:701– 7. [32] Bassaris HP, Gogos CA, Skoutelis AT, Starakis IK. Cytokines in sepsis: pathogenesis and therapeutic potential. Antibiot Clin 2003;7:181– 5. [33] Sawa T, Gorry DB, Gropper MA, Ohara M, Kurahashi K, WienerKronish JP. IL-10 improves lung injury and survival in Pseudomonas aeruginosa pneumonia. J Immunol 1997;159:2858–66. [34] Rubin JB, Pomeroy C. Role of gamma interferon in the pathogenesis of bacteremic pneumococcal pneumonia. Infect Immun 1997;65:2975–7. [35] Tessier PR, Kim M, Zhou W, et al. Pharmacodynamic assessment of clarithromycin in a murine model of pneumococcal pneumonia. Antimicrob Agents Chemother 2002;22:1425–34.