Recent Advances in the of in Children Magdy M.K. Mahmoud,1 Abdel-Raouf Omar,2 Gharib Fawy,3 and Khaled Salah4 From the Departments of Pediatrics,1 Neurology2,3 and Clinical Pathology,4 El-Minia1,2,4 and Sohag3 Universities

Abstract: Over the past decade, much has changed on the landscape of meningitis. The purpose of this study was to investigate the involvement of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) in the - pathogenesis of childhood meningitis. We measured the concentration of NO 2 (a stable metabolite of NO) and TNF-α in serial samples of (CSF) from 21 children with septic and 18 with - aseptic meningitis and 20 control patients without meningitis. Significantly higher CSF NO 2 concentrations were detected in those with bacterial meningitis than those with aseptic meningitis (27.6 ± 26.8 versus 12.2 ± 12.3 µmol/l ; P<0.001) or among non-meningitis subjects (13.2 ± 24.2 µmol/l; P<0.0001). Clinical and laboratory improvement following administration of antibiotics and - dexamethasone was associated with a fall in CSF (NO 2) to normal levels in these patients. The mean (±SD) of concentration in septic meningitis was 148.74 ± 338.77 pg/ml. There was significantly more TNF- alpha than aseptic meningitis (6.85 ± 17.93 pg/ml; P<0.001) or non-meningitis (7.67 ± 16.07 pg/ml; P<0.001). We did not find a correlation between CSF nitrate/nitrite levels and TNF-α (r = 0.046). Our findings indicate that NO and TNF-α production are enhanced in the CSF compartment of children with septic meningitis and support the hypothesis that both markers are involved in the pathophysiology of septic meningitis.

Abbreviations: NO: Nitric oxide, NOS: Nitric oxide synthases, TNF-α:Tumor Necrosis Factor Alpha.

Introduction: unstable molecule peroxynitrite (OONO-), while that between NO and thiol groups produces nitrosothiols. Nitric oxide (NO) is a labile free radical produced by a These reactive nitrogen intermediates inactivate key variety of cells and is involved in such physiologic microbial enzymes, such as ribonucleotide reductase processes as smooth muscle relaxation, neuronal and aconitase, by reacting with iron containing signaling, inhibition of platelet aggregation and groups in these enzymes.8,9 regulation of cell mediated cytotoxicity.1-3 NO is In bacterial meningitis, meningeal inflammation is formed by the oxidative deamination of the amino initiated by local production of proinflammatory acid L-arginine by nitric oxide synthases (NOS). cytokines, especially tumor necrosis factor alpha Three isoforms of this enzyme are described. (TNF-α) and interleukin 1β,10,11 which are both potent Neuronal NOS (nNOS or NOS1) is constitutively stimuli of iNOS expression in leucocytes, glia, and present in both the central and peripheral nervous neurons. Whether NO acts as a neurotoxic or systems, where NO acts as a neurotransmitter. neuroprotective molecule may be dependent on its Endothelial NOS (eNOS or NOS3) is constitutively redox state; in its oxidized form (NO+) inactivates expressed by endothelium and other cell types and is glutamate receptors and reduces the neurotoxicity of involved in cardiovascular homeostasis. In contrast, excitatory amino acids, whereas the reduced form inducible NOS (iNOS or NOS2) is absent in resting (NO-) can form peroxynitrate, a potent cells, but the gene is rapidly expressed in response to neurotoxin(5,7,9). The therapeutic use of NOS inhibitors stimuli such as proinflammatory cytokines. Once in bacterial meningitis has been suggested, but as present, iNOS synthesises 100–1000 times more NO NO is likely to have profound effects on cerebral than the constitutive enzymes and does so for blood flow and other important physiological prolonged periods. This high concentration of NO processes, our understanding of the role of NO in may inhibit a large variety of microbes, but may also meningitis seems inadequate to justify this potentially damage the host, thereby contributing to intervention at present.12-14 pathology.4-7 In this study, we investigated the role of NO The antimicrobial effect stems not from NO itself, but production in childhood meningitis by determining the - from reactive nitrogen intermediates formed by the levels of (NO 2) in cerebrospinal fluid samples. We oxidation of NO. Reaction between NO and the free also investigated the relationship between CSF - radical superoxide (O2 ) results in the formation of the nitrates/nitrite and TNF-alpha levels.

Alexandria Journal of Pediatrics, Volume 19, Number 1, January 2005 13 Subjects and Methods: Results: The study population consisted of 40 patients: 21 The cerebrospinal fluid characteristics of the studied children with septic meningitis (12 boys and 9 girls) groups (septic meningitis, aseptic meningitis and with an age range from 2 months to 8 years (mean ± controls) were illustrated in table I. SD, 3.2 ± 2.2 years) and 19 children with aseptic meningitis (11 boys and 8 girls) with an age range Table I: CSF characteristics of the studied groups; septic from 6 months to 7 years (2.6 ± 2.1 years). Septic meningitis, aseptic meningitis and controls Aseptic meningitis was diagnosed by the presence of positive Septic Controls meningitis meningitis bacterial cultures of CSF and blood, in addition to (n = 21) (n = 19) (n = 20) clinical manifestations of meningitis, highly elevated Age (years) 3.2 ± 2.2 2.6 ± 2.1 2.8 ± 1.9 CRP, and polymorphonuclear pleocytosis in CSF. Sex: - Boys 12 11 12 Children with septic meningitis were treated with - Girls 9 8 8 intravenous beta-lactam antibiotics (e.g. ampicilin, CSF: cefotaxime, ceftriaxone) with dexamethasone (0.6 - WBC/mm3 4010 ± 380 100 ± 2.03 0 mg/kg/day in 4 divided doses) at least for 2 weeks - (mg/dl) 24.6 ± 22.7 70.5 ± 11.2 50 ± 12 and were observed, clinically and laboratory, for - Protein (mg/dl) 264.3 ± 100.5 110.3 ± 21.4 42 ± 5.2 - TNF-α (pg/ml) 148.74 ± 338.77 6.85 ± 17.93 7.67 ± 16.07 another 2 weeks. All patient showed clinical and - NO--2 (µmol/l) 27.6 ± 26.8 12.2 ± 12.3 13.2 ± 24.2 laboratory response to the treatment. The diagnosis Significance P1 = 0.0001** P2 = 0.05* P3 = 0.0003** of aseptic meningitis depended on the clinical P4 = 0.001* P5 = 0.001* P6 = 0.001* manifestations, negative CRP, mononuclear P1: Comparison between CSF TNF-α levels in septic meningitis and controls. P2: Comparison between CSF TNF-α levels in aseptic meningitis and controls. pleocytosis in CSF, and negative bacterial cultures of P3: Comparison between CSF TNF-α levels in septic and aseptic meningitis. — CSF. P4: Comparison between CSF NO 2 levels in septic meningitis and controls. — P5: Comparison between CSF NO 2 levels in aseptic meningitis and controls. — The control group consisted of 20 children who P6: Comparison between CSF NO 2 levels in septic and aseptic meningitis. presented with fever that required analysis of CSF to * Significant, ** highly significant P value (< 0.05.) exclude the presence of meningitis. They were 12 - boys and 8 girls aged 2.8 ± 1.9 years (range from 3 The mean NO 2 in CSF samples obtained during the months to 6.5 years). Informed consent was obtained early stages of the disease in patients with septic from the parents of all patients enrolled in this study. meningitis (27.6 ± 26.8 µmol/l) was significantly CSF Samples were obtained on the first day of higher than control group (13.2 ± 24.2 µmol/l, admission and were kept at -70°C. Determination of p<0.001) (figures 1,2). nitrate/nitrite level was performed by use of the nitrate/nitrite Colorimetric Assay Kit (LDH Method) NO TNF- (Cayman Chemical, USA). This kit provided the measurement of total nitrate/nitrite concentration in 2- step process. The first step was the conversion of nitrate to nitrite by the enzyme nitrate reductase. The second step was the addition of Griess reagent, which converts nitrite into a deep purple azo compound. The absorbance of standards and samples was read at 540 nm. TNF-α levels were assayed in CSF by the use of an Septic meningitis Aseptic meningitis Controls ELISA kit (Quantikine, UK) according to the Septic meningitis Aseptic meningitis Control manufacture’s instruction. Figure 1: NO-2 and TNF- α in CSF samples obtained from control patients and patients with septic, and aseptic meningitis. Data are Statistical Analysis: means ± SD.

Results were expressed as mean ± SD. Statistical Following clinical and laboratory improvement, the - analysis was performed using unpaired and paired CSF NO 2 diminished to normal range (table II). The - two-tailed t test. Pearson’s correlation was used to CSF NO 2 in patients with aseptic meningitis remained measure how variables or rank orders are related. A p below the normal upper limit level throughout the value of <0.05 denoted the presence of statistical observation period (12.2 ± 12.3 µmol/l, P<0.001). significance.

Alex J Pediatr, 19 (1), Jan 2005 14 Table II: CSF levels of both NO-2 and TNF-α levels before and after treatment in patients with septic and aseptic meningitis and controls Septic meningitis Aseptic meningitis Before Treatment After Treatment p Before Treatment After Treatment p NO-2 (µmol/l) 27.6 ± 26 1.9 ± 0,6 S 12.2 ± 10 1.7 ± 0.4 NS TNF-α (pg/ml) 901 ± 101 287 ± 101 S 301 ± 201 211 ± 23s NS S = significant p value (< 0.05.), NS = Non-significant p value

completely understood. Recently, it has been Nitric Oxide (umol/l) 80 suggested that nitric oxide (NO), a labile free radical,

70 contributes to pathological processes in bacterial meningitis.7,9,11 60     -   In our study, we found that the CSF NO 2 level in 50   children with septic meningitis before treatment was 40        significantly higher than that in aseptic meningitis and 30             the controls, while after clinical and laboratory      20        improvement the CSF NO- level was insignificantly     2 10       differ in the three groups of the study. These results 0 Septic mening. Aseptic mening. Control indicate that the septic meningitis in children leads to Figure 2: CSF levels of nitric oxide (NO) in controls and patients with septic increase production of NO in CSF and the fall in its and aseptic meningitis level after response to treatment is another evidence for this effect. In contrast, children with aseptic - meningitis did not show high NO 2 concentrations, Tumor necrosis f actor-alpha (pg/ml) 180 suggesting that viral factors do not cause a 170 substantial induction of NO production in children. 160  150   Our observations were in agreement with those of 140    130   Korneliss et al.,15 in 1996, Uysal et al.,16 in 1999, 120  110    Murawaska et al.,17 in 2000 and Azumagaura et al.,18 100   90  in 2003, who reported a significantly high levels of 80   - 70 NO 2 in CSF samples obtained from children during 60 50      the early stages of septic meningitis support the 40   30    presence of an enhanced production of NO in the 20      1 10        CSF compartment. Milstein et al., in 1994 and 0   19 Septic mening. Aseptic mening. Control Hockett et al., in 1995, reported increased CSF Figure 3: CSF levels of tumor necrosis alpha in controls and patients with levels of NO metabolites in adult patients with septic and aseptic meningitis bacterial meningitis. NO synthesis may be immuno-induced by bacterial The mean (± SD) of TNF-α concentration in septic lipopoly- saccharides and inflammatory cytokines in a meningitis was 148.74 ± 338.77 pg/ml. There was variety of cells, including neutrophils, significantly more TNF-α than aseptic meningitis microglia/macrophages, microvascular endothelial (6.85 ± 17.93 pg/ml; P<0.0003) or non-meningitis cells, astrocytes and neuronal cells in the CSF (7.67 ± 16.07 pg/ml; P<0.0001)(figures 1,3). Mean compartment.17,18 CSF nitrate/nitrite levels did not correlate with TNF-α The mechanism by which high levels of NO (r = 0.046), white blood count (r = 0.217), or protein contribute to the blood-brain and blood-CSF barrier (r = 0.263). However, there was a moderate negative permeability changes is not fully understood. One correlation with CSF glucose levels (r = -0.46, p = plausible scenario is that excessive levels of NO 0.056). disrupt various enzyme systems associated with Discussion: mitochondrial respiration, DNA replication and citric acid cycle in microvascular endothelial cells, thereby Bacterial meningitis continues to be an important causing cellular destruction or alteration and loss of cause of morbidity and mortality during infancy and integrity of these barriers.5,7,9 Also, we found that the childhood despite the availability of effective CSF level of TNF-α was significantly higher in septic bactericidal antibiotics. Although important advances meningitis than in aseptic meningitis and the controls. have been made in clarifying the pathophysiological Our results were in accordance with those reported events in meningitis, the mechanism responsible for by others.16,19,20 damage of central nervous system are not yet

15 Alex J Pediatr, 19 (1), Jan 2005 In contrast to the previous data,4,10 we found no decreased CSF glucose concentration in patients correlation between nitrate/nitrite and TNF-α levels of with bacterial meningitis. In addition, inhibition of CSF. The main product of NO in CSF is nitrite and carrier-mediated transport across the blood-brain this may be a cause for the absence of a correlation barrier causes low CSF glucose levels. with TNF-α level.16 We found no correlation between CSF nitrate/nitrite levels and CSF white blood count Conclusion: or protein levels. There was, however, a moderate Our observations indicate that NO and TNF-α negative correlation with CSF glucose levels, production is enhanced in the CSF compartment of confirming previously reported data.4,16 NO children with septic meningitis and support the suppresses cellular oxidative metabolism through the hypothesis that both markers contribute to the inhibition of tricarboxylic acid cycle and electron pathophysiology of septic meningitis. transport chain enzyme and enhances anaerobic .21-23 This mechanism may contribute to the

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