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    • Journal of Antimicrobial Chemotherapy (2003) 52, 651–655 DOI: 10.1093/jac/dkg417 Advance Access publication 1 September 2003 651 .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. © The British Society for Antimicrobial Chemotherapy 2003; all rights reserved. Factors influencing the anti-inflammatory effect of dexamethasone therapy in experimental pneumococcal meningitis I. Lutsar*, I. R. Friedland, H. S. Jafri, L. Wubbel, A. Ahmed, M. Trujillo, C. C. McCoig and G. H. McCracken Jr Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA Received 11 January 2003; returned 24 February 2003; revised 14 July 2003; accepted 15 July 2003 Dexamethasone (DXM) interferes with the production of tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1) and can thereby diminish the secondary inflammatory response that follows initiation of antibacterial therapy. A beneficial effect on the outcome of Haemophilus meningitis in children has been proven, but until recently the effect of DXM therapy in pneumococcal meningitis was uncertain. The aim of the present study was to evaluate factors that might influence the modulatory effect of DXM on the antibiotic-induced inflam- matory response in a rabbit model of pneumococcal meningitis. DXM (1 mg/kg) was given intravenously 30 min before or 1 h after administration of a pneumococcal cell wall extract, or the first dose of ampicillin. In meningitis induced by cell wall extract, DXM therapy prevented the increase in cerebrospinal fluid (CSF) leucocyte and lactate concentrations, but only if given 30 min before the cell wall extract. In meningitis caused by live organisms, initiation of ampicillin therapy resulted in an increase in CSF TNF-α and lactate concentrations only in animals with initial CSF bacterial concentrations ≥5.6 log10 cfu/mL. In those animals, DXM therapy prevented significant elevations in CSF TNF-α [medianchange–184pg/mL,–114pg/mLversus +683 pg/mL with DXM (30 min before or 1 h after ampicillin) versus controls (no DXM), respectively, P = 0.02] and lactate concentrations [median change –10.6 mmol/L, –1.5 mmol/L versus +14.3 mmol/L with DXM (30 min before or 1 h after ampicillin) versus controls (no DXM), respectively, P = 0.01]. These effects were inde- pendent of the timing of DXM administration. In this model of experimental pneumococcal meningitis, an antibiotic-induced secondary inflammatory response in the CSF was demonstrated only in animals with high initial CSF bacterial concentrations (≥5.6 log10 cfu/mL). These effects were modulated by DXM therapy whether it was given 30 min before or 1 h after the first dose of ampicillin. Keywords: animal models, CSF, experimental meningitis, inflammatory response, S. pneumoniae Introduction Pneumococcal meningitis remains a significant cause of morbidity and mortality. It is the host’s own inflammatory response that is responsible for the central nervous system injury characteristic of the disease.1This inflammatory response can be exacerbated by antibac- terial therapy that increases rapidly the release of proinflammatory pneumococcal cell wall products.2–5 Currently, dexamethasone (DXM) is the only anti-inflammatory agent that has been documented to improve the outcome of bacterial meningitis in clinical trials, most convincingly in children with Hae- mophilus influenzae meningitis.6 DXM inhibits production of TNF-α and IL-1, reverses development of brain oedema and limits the increase in cerebrospinal fluid (CSF) lactate and leucocyte concen- trations.1,7,8 Studies have suggested that in H. influenzae meningitis the timing of DXM in relation to the first antibiotic injection is critical. The antibiotic-induced inflammatory response is prevented only if DXM therapy is given simultaneously with ceftriaxone; DXM given 1 h later failed to modulate the antibiotic-induced inflammatory response.3 A meta-analysis of 10 clinical trials in children suggested that the timing of DXM therapy might also be critical in pneumo- coccal meningitis. DXM therapy prevented the development of severe hearing loss only when it was given before or at the same time as the first antibiotic injection (OR = 0.09; 95% CI: 0.00–0.71).6 Recently de Gans & van de Beek9 in a prospective, randomized, placebo-controlled, multicentre trial demonstrated the beneficial effect of adjunctive DXM therapy on the outcome of pneumococcal meningitis in adults. The present study was conducted to evaluate the modulatory effect of DXM therapy given 30 min before, compared with 1 h after, .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . . . . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. .. **Correspondence address. Clinical Development, Pfizer Ltd., Ramsgate Road ,CT13 9NJ, UK. Tel: +44-1304-645173; Fax:+44-1304-655669; E-mail: irja_lutsar@sandwich.pfizer.com byguestonFebruary19,2012http://jac.oxfordjournals.org/Downloadedfrom
    • I. Lutsar et al. 652 intracisternal injection of pneumococcal cell walls, or after the first injection of ampicillin, in experimental meningitis caused by live pneumococci. Material and methods Inocula Pneumococcal cell wall (1 cm3 of this product was obtained from 2.5 × 109 live organisms) was produced and provided by Dr Elaine Tuomanen.10 Streptococcus pneumoniae (MIC and MBC of ampicillin, 0.01 mg/L) was originally isolated from a patient with bacterial menin- gitis. To induce meningitis, 0.2 mL pneumococcal cell wall product, or 104–105 cfu/mL oflive organisms, were inoculated intracisternally. Meningitis model and treatment A rabbit meningitis model originally described by Dacey & Sande11 was used. In the first set of experiments, DXM (1 mg/kg) was given intravenously 30 min before or 1 h after the administration of pneumo- coccal cell walls to six and seven animals, respectively. In the second set ofexperiments, 16–18 h afterinoculation ofliveorganisms,animalswere treated with ampicillin alone (75 mg/kg every 12 h) for 24 h, or with the combinationofampicillinand intravenousDXM(1mg/kg)given30min before or 1 h after the first ampicillin dose. Each treatment group con- sisted of 12–13 animals. No treatment was given to control animals. CSF sample collection and analysis CSF was collected directly from the cisterna magna under acepromazine and ketamine anaesthesia before and 2, 4, 6, 12 and 24 h after start of therapy. For the first four CSF collections, animals were restrained under anaesthesia in stereotactic frames. Leucocytes were counted in a Neubauer haemocytometer. Bacterial concentrations in CSF were measured by plating undiluted and serial dilutions of CSF on sheep blood agar and incubating in 5% CO2 at 35°C for 24 h. The lower limit of detection was 10 cfu/mL. The remaining CSF was centrifuged and the supernatant stored at –70°C. CSF lactate concentrations were meas- ured by a photocolorimetric assay (Behring Diagnostics Inc, Milton Keynes, UK). TNF-α concentrations were measured by cytotoxic assay using L929 tumorigenic murine fibroblasts.12 The standard curve for theTNF-α assay was linearfrom 40–2500 pg/mL. Statistical analysis Normallydistributeddataarepresentedas mean ±S.D. andnon-normally distributed data as median and range. Student’s t-test was used for comparison of parametric data and the Kruskall–Wallis analysis of variance for non-parametric variables. Results Meningitis induced by pneumococcal cell walls Administration of pneumococcal cell walls resulted in the release of TNF-α, an influx of leucocytes and increased lactate concentrations in CSF (Figure 1). The elevation in TNF-α concentrations was pre- vented by DXM therapy when given 30 min before or 1 h after pneu- mococcal cell walls. The increase in CSF leucocyte and lactate concentrations,however,wasinhibitedonlywhenDXMtherapywas given 30 minbefore the cell wall products. Meningitis induced by live organisms The addition of DXM to ampicillin therapy resulted in a lower initial bacterial killing rate compared with ampicillin therapy alone (0.39 ± 0.1 cfu/mL/h versus 0.57 ± 0.12 cfu/mL/h; P = 0.04). The changes in CSF inflammatory indices were similar in all study groups and were not influenced bythe co-administrationofDXM(Figure 2). For further analysis, animals were divided in two groups based on CSF bacterialconcentrationsat the start oftherapy(≤5.5log10 or≥5.6 log10 cfu/mL) (Figure 3). After the first dose of ampicillin, animals with high initial bacterial concentrations demonstrated significantly greater changes in CSF TNF-α [median ∆+683 pg/mL (quartiles +246 to +758) versus ∆–16.3 pg/mL (quartiles –6 to –29)], white bloodcells (WBC)[median ∆+2175 cells/mL (quartiles 437to 6987) versus ∆–325 cells/mL (quartiles –787 to +25)] and lactate [median ∆+14.3 mmol/L (quartiles –7.6 to –14.6) versus ∆–8.5 mmol/L (quartiles–5.1to–16.4)] concentrationscomparedwithanimalswith lowerinitial bacterial concentrations. Figure 1. Concentrations of WBC (cells/mL; left), TNF-α (pg/mL; middle) and lactate (mmol/L; right) in the CSF. Meningitis was induced by the intracisternal administration ofpneumococcalcellwalls.Animalsweretreated with DXMgiven 30 min before (open squares)or 1h after (solid squares) administration of cellwalls. No treatment was given to the control animals (solid triangles). *P = 0.02 versus controls or those treated with DXM 1 h after introduction of cell walls. byguestonFebruary19,2012http://jac.oxfordjournals.org/Downloadedfrom
    • Dexamethasone in pneumococcal meningitis 653 In those with high initial bacterial concentrations (≥5.6 log10 cfu/ mL), DXM therapy prevented elevations in CSF TNF-α [median ∆–184 pg/mL (quartiles –116 to –258) or ∆–114 pg/mL (quartiles – 39 to –175) versus ∆+683 pg/mL (quartiles 246 to +758) with DXM given 30 min before or 1 h after ampicillin versus without DXM, respectively; P = 0.02] and lactate concentrations [median ∆–10.6 mmol/L (quartiles 7.6 to 17.4) or ∆–1.5 mmol/L (quartiles –19.7 to – 1.3) versus ∆+14.3 mmol/L (quartiles –7.6 to –14.6) with DXM given 30 min before or 1 h after ampicillin and without DXM, respectively; P = 0.01]. These effects were not significantly different as a result of the timing of DXM administration, although there was a trend indicating that changes in TNF-αand lactate values were lower in animals given DXM before ampicillin therapy (Figure 4). The changes in leucocyte concentrations were not affected by DXM ther- apy. In animals with low CSF bacterial concentrations (≤5.5 log10 cfu/mL), there were no differences in TNF-α, WBC or lactate con- centrations between those treated with or without DXM (data not shown). There was no correlation between changes in TNF-α, leuco- cyte and lactate concentrations in CSF, and the degree of bacterial killing after introduction of ampicillin therapy (r = 0.06; r = 0.47 and r =0.46, respectively; P> 0.05). Discussion In this model of pneumococcal meningitis, we demonstrated that the antibiotic-induced secondary inflammatory response, as evidenced Figure 2. Concentrations of WBC (cells/mL), TNF-α (pg/mL), lactate (mmol/ L) and S. pneumoniae (log10 cfu/mL) in CSF in animals with pneumococcal meningitis. Animals were treated with ampicillin alone (solid triangles), with DXM given 30 min before (open squares) or 1 h after ampicillin therapy (solid squares). No treatment was given to the control (open triangles) animals, and they died after 6–10 h. Error bars demonstrate lower and upper quartile. *P = 0.04 versus those not receiving DXM therapy. Figure 4. Concentrations of WBC (cells/mL, left), TNF-α (pg/mL, middle), lactate (mmol/L, right) in CSF in animals with initial CSF bacterial concentrations ≥5.6 log10 cfu/mL. DXM therapy was given 30 min before (open squares) or 1 h after the first dose of ampicillin (solid squares). Control animals (solid triangles) were treated with ampicillin only. Error bars demonstrate lower and upper quartiles. *P < 0.05 versus those treated with ampicillin and DXM. Figure 3. Changes in CSF concentrations of WBC (cells/mL), TNF-α (pg/mL), lactate (mmol/L) and S. pneumoniae (log10 cfu/mL) in pneumococcal meningi- tis after introduction of ampicillin therapy. The CSF bacterial concentrations at the introduction of ampicillin therapy were ≤5.5 log10 cfu/mL (open squares) or ≥5.6 log10 cfu/mL (solid squares). Error bars demonstrate lower and upper quar- tile. *P < 0.05 byguestonFebruary19,2012http://jac.oxfordjournals.org/Downloadedfrom
    • I. Lutsar et al. 654 by an elevation of CSF TNF-α and lactate concentrations, occurred only in animals with high bacterial concentrations before the start of ampicillin therapy. The increase in CSF lactate and TNF-α con- centrations were inhibited by adjunctive DXM therapy regardless of whether it was given 30 min before or 1 h after the first dose of ampicillin. Liberation of free endotoxin or cell-wall components by cell-wall active antibiotics has been demonstrated in vitro and in experimental meningitis.3,4,5,13 This is associated with enhanced inflammation in the subarachnoid space, as evidenced by an increase in leucocyte, TNF-αand lactate concentrations.3,14 Ourstudyshowed that the anti- biotic-induced inflammatory burst did not occur in all animals and was seen only in those with greater CSF bacterial concentrations (≥5.6 log10 cfu/mL). Although this has been intimated previously, data were not provided.14 In experimental meningitis, the magnitude oftheinflammatoryresponseinCSFdepends ontheconcentrationof inoculated cell walls10 and thus it is not surprising that animals with higher bacterial counts demonstrate a more pronounced host inflam- matory response after antibiotic therapy. There is concern that rapid bacterial killing by antibiotics could result in an enhanced inflammatory response and a worsening of the clinical outcome in meningitis.15,16 The results of this and previous studies, however, do not support these speculations. On the contrary, clinical and experimental studies have demonstrated that rapid effec- tive bactericidal therapy protects against the development of deaf- ness and other neurological disabilities.17,18 Moreover, this and previous studies4 found no correlation between CSF inflammatory markers and the magnitude of bacterial killing. Uncontrolled bac- terial growth eventually results in a much greater release of cell wall components—and consequent enhancement of the inflammatory response—than that induced by antibacterial therapy.4,13 These find- ings suggest that early antibacterial therapy and rapid clearance of bacteria from CSF outweigh the potential adverse effects caused by the antibiotic-induced inflammatoryburst. DXM, as an adjunct to antibacterial therapy in bacterial meningi- tis, has been shown to be beneficial in H. influenzae meningitis in children.6 In pneumococcal meningitis, however, its modulatory effect was uncertain because of the relatively small numbers of chil- dren enrolled in prospective controlled trials.19 In adults with pneu- mococcal meningitis, an unfavourable outcome was seen in 26% of patientsreceivingadjunctiveDXMcomparedwith52%amongthose receiving placebo.9 The results of our study clearly supported previ- ous findingsbyTuomanenet al.8 and showed that,similarto H. influ- enzae meningitis, DXM therapy prevents the antibiotic-induced release ofTNF-αand lactateconcentrationsinCSF inpneumococcal meningitis.3 Early institution of DXM therapy has been suggested because of its delayed onset of action.20 In our study, the timing of DXM appeared to be critical only in meningitis induced by the pneumo- coccal cell wall. Inmeningitis induced bylive microorganisms, how- ever, once CSF inflammation was established, administration of DXM before the dose of ampicillin did not appear to have a signifi- cantlygreater salutaryeffectthan1hafter ampicillin;DXM adminis- tration that was delayed further was not assessed. These results contrast with those obtained in experimental H. influenzae meningi- tis, where the antibiotic-induced inflammatory response was modu- lated only if DXM was given before or simultaneously with ceftriaxonetherapy.3 The effectivenessofearly DXM administration was also highlighted ina recent meta-analysis of 10 randomized con- trolled clinical trials.6 At the time of diagnosis, ∼40% of patients with pneumococcal meningitishave CSFbacterial concentrations<106 cfu/mL21,andour findings suggest that antibiotic-inducedenhancedinflammationmay not occur in such patients. Whether these patients benefit from DXM therapy, and how they can be identified at diagnosis, requires further clarification. Detection of bacteria in Gram-stained specimens of CSF maybeuseful inidentifyingpatientswithlarge bacterialloads.22 One possible detrimental effect of DXM therapy, also demon- strated in this study, is decreased bacterial clearance from CSF.23,24 DXMdecreasesthepermeabilityoftheblood–brainbarrier,resulting in decreased concentrations of hydrophilic antibiotics in the CSF.25 Also,highconcentrationsofDXM(400 µg/mL)inhibitphagocytosis by CSF leucocytes.26The clinical relevance of these findings, how- ever, has not been demonstrated.6,9 In this model of pneumococcal meningitis, CSF bacterial concen- trations at the start of therapy appeared to be more important than the timing ofDXMtherapy in influencing the antibiotic-induced inflam- matory response. It is likely that there is a time beyond which DXM loses its effectiveness, but this point has not been clearly defined. Acknowledgements I. Lutsar was a recipient of a fellowship award from the European Society for Paediatric Infectious Diseases, supported by Lederle- PraxisBiologicals.Partofthisstudywaspresentedatthe1997annual meeting of Infectious Diseases Society of America (IDSA), San Francisco. The study has followed animal experimentation guide- lines and was approved by the Institutional Animal Care and Research Advisory Committee of the University ofTexas. References 1. Spellerberg, B. & Tuomanen, E. I. (1994). Pathophysiology of pneumococcal meningitis. Annals of Medicine 26, 411–18. 2. Nau, R., Zysk, G., Schmidt, H. et al. (1997). Trovafloxacin delays the antibiotic-induced inflammatory response in experimental pneumo- coccal meningitis. Journal of Antimicrobial Chemotherapy 39, 781–8. 3. Mustafa, M. M., Ramilo, O., Mertsola, J. et al. (1989). Modulation of inflammation and cachectin activity in relation to treatment of experimen- tal Haemophilus influenzae type b meningitis. Journal of Infectious Diseases 160, 818–25. 4. Friedland, I. R., Jafari, H., Ehrett, S. et al. (1993). Comparison of endotoxin release by different antimicrobial agents and the effect on inflammation in experimental E. coli meningitis. Journal of Infectious Diseases 168, 657–62. 5. Fischer, H. & Tomasz, A. (1984). Production and release of pepti- doglycan and wall teichoic acid polymers in pneumococci treated with beta lactam antibiotics. Journal of Bacteriology 157, 507–13. 6. McIntyre, P. B., Barkey, C. S., King, S. M. et al. (1997). Dexame- thasone as adjunctive therapy in bacterial meningitis: a meta-analysis of randomized clinical trials since 1988. Journal of the American Medical Association 278, 925–31. 7. Täuber, M. G., Khayam-Bashi, H. & Sande, M. A. (1985). Effects of ampicillin and corticosteroids on brain water content, cerebrospinal fluid pressure, and cerebrospinal fluid lactate levels in experimental pneumococcal meningitis. Journal of Infectious Diseases 151, 528–34. 8. Tuomanen, E., Hengstler, B., Rich, R. et al. (1987). Nonsteroidal anti-inflammatory agents in the therapy for experimental pneumococcal meningitis. Journal of Infectious Diseases 155, 985–90. 9. de Gans, J. & van de Beek, D. (2002). Dexamethasone in adults with bacterial meningitis. New England Journal of Medicine 347, 1549–56. 10. Tuomanen, E., Liu, H., Hengstler, B. et al. (1985). The induction of meningeal inflammation by components of the pneumococcal cell wall. Journal of Infectious Diseases 151, 859–68. byguestonFebruary19,2012http://jac.oxfordjournals.org/Downloadedfrom
    • Dexamethasone in pneumococcal meningitis 655 11. Dacey, R. G. & Sande, M. A. (1974). Effect of probenecid on cerebrospinal fluid concentration of penicillin and cephalosporin derivates. Antimicrobial Agents and Chemotherapy 6, 437–41. 12. Flick, D. A. & Gifford, G. E. (1984). Comparison of in vitro cell cyto- toxic assays for tumor necrosis factor. Journal of Immunological Methods 68, 167–75. 13. Stuertz, K., Schmidt, H., Eiffert, H. et al. (1998). Differential release of lipoteichoic and teichoic acids from Streptococcus pneumo- niae as a result of exposure to β-lactam antibiotics, rifamycins, trova- floxacin, and quinupristin–dalfopristin. Antimicrobial Agents and Chemotherapy 42, 277–81. 14. Pfister, H.-W., Fontana, A., Täuber, M. G. et al. (1994). Mecha- nism of brain injury in bacterial meningitis: workshop summary. Clinical Infectious Diseases 19, 463–79. 15. Tuomanen, E. I., Austrian, R. & Masure, H. R. (1995). Pathogene- sis of pneumococcal infection. New England Journal of Medicine 332, 1280–4. 16. Täuber, M. G., Shibl, A. M., Hackbarth, C. J. et al. (1987). Anti- biotic therapy, endotoxin concentration in cerebrospinal fluid, and brain edema in experimental Escherichia coli meningitis in rabbits. Journal of Infectious Diseases 156, 456–62. 17. Winter, A. J., Comis, S. D., Osborne, M. P. et al. (1998). Ototoxicity resulting from intracochlear perfusion of Streptococcus pneumoniae in the guinea pig is modified by cefotaxime or amoxycillin pretreatment. Journal of Infection 36, 73–7. 18. Lebel, M. H. & McCracken, G. H., Jr (1989). Delayed cerebro- spinal fluid sterilization and adverse outcome of bacterial meningitis in infants and children. Pediatrics 83, 161–7. 19. Arditi, M., Mason, E. O., Bradley, J. S. et al. (1998). Three-year multicenter surveillance of pneumococcal meningitis in children: clinical characteristics, and outcome related to penicillin susceptibility and dexa- methasone use. Pediatrics 102, 1087–97. 20. Nakamura, H., Mizushima, Y., Seto, Y. et al. (1985). Dexametha- sone fails to produce antipyretic and analgesic actions in experimental animals. Agents and Actions 16, 542–7. 21. Bingen, E., Lambert-Zechovsky, N., Mariani-Kurkdjian, P. et al. (1990). Bacterial counts in cerebrospinal fluid of children with meningitis. European Journal of Clinical Microbiology and Infectious Diseases 9, 278–81. 22. Feldman, W. E., Ginsburg, C. M., McCracken, G. H., Jr et al. (1982). Relation of concentrations of Haemophilus influenzae type b in cerebrospinal fluid to late sequelae of patients with meningitis. Journal of Pediatrics 100, 209–12. 23. Schaad, U. B., Kaplan, S. L. & McCracken, G. H. (1994). Steroid therapy for bacterial meningitis. Clinical Infectious Diseases 20, 685–90. 24. Cabellos, C., Martinez-Lacasa, J., Tubau, F. et al. (2000). Evalua- tion of combined ceftriaxone and dexamethasone therapy in experimental cephalosporin-resistant pneumococcal meningitis. Journal of Antimicrobial Chemotherapy 45, 315–20. 25. Lutsar, I., McCracken, G. H. & Friedland, I. R. (1998). Antibiotic pharmacodynamics in cerebrospinal fluid. Clinical Infectious Diseases 27, 1117–29. 26. Weitbrecht, W. U. (1980). Influence of milieu and dexamethasone on in vitro phagocytosis of cerebrospinal fluid phagocytes. Pathological Research and Practice 167, 393–9. byguestonFebruary19,2012http://jac.oxfordjournals.org/Downloadedfrom