Effects of B7-H3 on the inflammatory response and expression of MMP-9 in mice with pneumococcal meningitis.

B7-H3, a new member of the B7 superfamily, plays a key role in the regulation of T cell-mediated immune responses. Our previous work showed that B7-H3 strongly augmented both LPS- and bacterial lipoprotein-induced NF-κB activation and inflammatory response, and soluble B7-H3 was elevated in CSF and plasma of patients with bacterial meningitis. MMP-9 has been implicated in blood-brain barrier disruption, inflammation, and vasculitis during the pathogenesis of bacterial meningitis. In this study, we report that in a murine model of pneumococcal meningitis, B7-H3 treatment enhances inflammatory response in the meninges, upregulates MMP-9 expression in cerebral parenchyma, and deteriorates clinical disease status indicated by weight loss and impaired movement ability. In vitro results showed that B7-H3 augmented MMP-9 secretion from Streptococcus pneumoniae-stimulated microglia cells. Thus, our data indicate that B7-H3 contributes to the development of pneumococcal meningitis by exaggerating inflammatory responses and upregulating MMP-9 activity in CNS, which ultimately lead to neuronal injury.

Identification of a novel pneumococcal vaccine antigen preferentially expressed during meningitis in mice.

Streptococcus pneumoniae is the most common cause of severe bacterial meningitis in children, the elderly, and immunocompromised individuals. To identify virulence factors preferentially expressed during meningitis, we conducted niche-specific genome-wide in vivo transcriptomic analysis after intranasal infection of mice with serotype 4 or 6A pneumococci. The expression of 34 bacterial genes was substantially altered in brain tissue of mice infected with either of the 2 strains. Ten upregulated genes were common to both strains, 7 of which were evaluated for their role in the development of meningitis. One previously uncharacterized protein, α-glycerophosphate oxidase (GlpO), was cytotoxic for human brain microvascular endothelial cells (HBMECs) via generation of H(2)O(2). A glpO deletion mutant was defective in adherence to HBMECs in vitro as well as in progression from the blood to the brain in vivo. Mutant bacteria also induced markedly reduced meningeal inflammation and brain pathology compared with wild type, despite similar levels of bacteremia. Immunization of mice with GlpO protected against invasive pneumococcal disease and provided additive protection when formulated with pneumolysin toxoid. Our results provide the basis of a strategy that can be adapted to identify genes that contribute to the development of meningitis caused by other pathogens.

The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release.

Streptococcus pneumoniae meningitis causes brain damage through inflammation-related pathways whose identity and mechanisms of action are yet unclear. We previously identified caspase-1, which activates precursor IL-1 type cytokines, as a central mediator of inflammation in pneumococcal meningitis. In this study, we demonstrate that lack of the inflammasome components ASC or NLRP3 that are centrally involved in caspase-1 activation decreases scores of clinical and histological disease severity as well as brain inflammation in murine pneumococcal meningitis. Using specific inhibitors (anakinra and rIL-18-binding protein), we further show that ASC- and NLRP3-dependent pathologic alterations are solely related to secretion of both IL-1ß and IL-18. Moreover, using differentiated human THP-1 cells, we demonstrate that the pneumococcal pore-forming toxin pneumolysin is a key inducer of IL-1ß expression and inflammasome activation upon pneumococcal challenge. The latter depends on the release of ATP, lysosomal destabilization (but not disruption), and cathepsin B activation. The in vivo importance of this pathway is supported by our observation that the lack of pneumolysin and cathepsin B inhibition is associated with a better clinical course and less brain inflammation in murine pneumococcal meningitis. Collectively, our study indicates a central role of the NLRP3 inflammasome in the pathology of pneumococcal meningitis. Thus, interference with inflammasome activation might be a promising target for adjunctive therapy of this disease.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis.

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

Brain creatine kinase activity after meningitis induced by Streptococcus pneumoniae.

Bacterial meningitis due to Streptococcus pneumoniae is associated with a significant mortality rate and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and memory. Creatine kinase (CK) is an effective buffering system of cellular ATP levels in high-energy consuming tissues; a decrease in CK activity is associated with a neurodegenerative pathway that results in neuronal loss. Thus, the aim of this study was to evaluate brain CK activity after pneumococcal meningitis. The animals underwent a magna cistern tap receiving either sterile saline as a placebo or an equivalent volume of a S. pneumoniae suspension; they were killed 6, 12, 24 and 48h after that, the brain was removed and hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used for the determination of CK activity. We verified that CK activity was not altered 6 and 12h after meningitis. Interestingly, 24h after the induction of the meningitis we observed a decrease in CK activity. Finally, CK activity was not altered 48h after meningitis. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity may be involved in the pathogenesis of pneumococcal meningitis.

Inducible nitric oxide synthase mediates hippocampal caspase-3 activation in pneumococcal meningitis.

Brain damage in bacterial meningitis is still a major problem. More knowledge about the triggers and mechanisms of neuronal damage in bacterial meningitis is needed to improve outcome in bacterial meningitis. The most common bacterial meningitis pathogen--Streptococcus pneumoniae--causes caspase activation and neuronal apoptosis in the hippocampus via its toxins and extensive inflammatory potential. Nitric oxide (NO)--produced by inducible nitric oxide synthase (iNOS)--is a major inflammatory mediator clearly upregulated in the cerebrospinal fluid during pneumococcal meningitis. However, its effects in bacterial meningitis are still controversial. This article demonstrates that genetic inactivation of iNOS results in a marked reduction of caspase-3-mediated neuronal damage in experimental murine pneumococcal meningitis. Protection of hippocampal neurons in iNOS knockout mice was not due to differences in intrathecal growth of S. pneumoniae and must therefore be attributed to differences of host inflammatory mediators. This indicates that NO plays an important role in hippocampal caspase-3 activation during pneumococcal meningitis.

JNK is activated but does not mediate hippocampal neuronal apoptosis in experimental neonatal pneumococcal meningitis.

Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.

Myeloid Src kinases regulate phagocytosis and oxidative burst in pneumococcal meningitis by activating NADPH oxidase.

Myeloid cells, including neutrophils and macrophages, play important roles in innate immune defense against acute bacterial infections. Myeloid Src family kinases (SFKs) p59/61(hck) (Hck), p58(c-fgr) (Fgr), and p53/56(lyn) (Lyn) are known to control integrin beta(2) signal transduction and FcgammaR-mediated phagocytosis in leukocytes. In this study, we show that leukocyte recruitment into the cerebrospinal fluid space and bacterial clearance is hampered in mice deficient in all three myeloid SFKs (hck(-/-)fgr(-/-)lyn(-/-)) during pneumococcal meningitis. As a result, the hck(-/-)fgr(-/-)lyn(-/-) mice developed increased intracranial pressure and a worse clinical outcome (increased neurologic deficits and mortality) compared with wild-type mice. Impaired bacterial killing was associated with a lack of phagocytosis and superoxide production in triple knockout neutrophils. Moreover, in hck(-/-)fgr(-/-)lyn(-/-) neutrophils, phosphorylation of p40(phox) was absent in response to pneumococcal stimulation, indicating a defect in NAPDH oxidase activation. Mice lacking the complement receptor 3 (CR3; CD11b/CD18), which belongs to the beta(2)-integrin family, also displayed impaired host defense against pneumococci, along with defective neutrophil superoxide production, but cerebrospinal fluid pleocytosis was normal. Cerebral expression of cytokines and chemokines was not decreased in both mouse strains, indicating that CR3 and myeloid SFKs are dispensable for the production of inflammatory mediators. Thus, our study demonstrates the pivotal role of myeloid SFKs and CR3 in mounting an effective defense against CNS infection with Streptococcus pneumonia by regulating phagocytosis and NADPH oxidase-dependent superoxide production. These data support the role of SFKs as critical mediators of CR3 signal transduction in host defense.

Increased expression of tyrosine phosphatase SHP2 in experimental pneumococcal meningitis: correlation with tumor necrosis factor-alpha and cerebrospinal fluid pleocytosis.

Protein tyrosine phosphatase SHP2 plays a crucial role in the development of the central nervous system. To explore the expression and possible role of SHP2 during the course of bacterial meningitis, this article reports a juvenile rat bacterial meningitis model established by direct intracisternal injection of Streptococcus pneumoniae. Expression of SHP2 at both mRNA and protein levels were assessed. White blood cell count and concentration of tumor necrosis factor-alpha (TNF-alpha) in cerebrospinal fluid (CSF) were also measured. In the cortex, bacterial meningitis led to a significant upregulation of mRNA encoding SHP2. SHP2 protein levels and CSF white blood cell count were positively correlated. However, there was no significant correlation between the levels of SHP2 protein and TNF-alpha concentrations in CSF. These findings do not support an essential role of SHP2 in the pathogenesis of experimental pneumoniae meningitis, but it is possible that SHP2 protein expression may be used as a marker of disease activity.

Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/- mice with experimental pneumococcal meningitis.

BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.

Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage.

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.

Differential regulation of blood-brain barrier permeability in brain trauma and pneumococcal meningitis-role of Src kinases.

Increased vascular permeability causing vasogenic brain edema is characteristic for many acute neurological diseases such as stroke, brain trauma, and meningitis. Src family kinases, especially c-Src, play an important role in regulating blood-brain barrier permeability in response to VEGF, but also mediate leukocyte function and cytokine signalling. Here we demonstrate that pharmacological inhibition of Src or c-Src deficiency does not influence cerebrospinal fluid (CSF) pleocytosis, brain edema formation, and bacterial outgrowth during experimental pneumococcal meningitis despite the increased cerebral expression of inflammatory chemokines, such as IL-6, CCL-9, CXCL-1, CXCL-2 and G-CSF as determined by protein array analysis. In contrast, inhibition of Src significantly reduced brain edema formation, lesion volume, and clinical worsening in cold-induced brain injury without decreasing cytokine/chemokine expression. While brain trauma was associated with increased cerebral VEGF formation, VEGF levels significantly declined during pneumococcal meningitis. Therefore, we conclude that in brain trauma blood-brain barrier tightness is regulated by the VEGF/Src pathway whereas c-Src does not influence brain edema formation and leukocyte function during bacterial meningitis.

Persisting vasculitis after pneumococcal meningitis.

INTRODUCTION: Bacterial meningitis is associated with a high mortality and a high incidence of neurological sequelae. Parainfectious vasculitis leading to ischemic brain damage is a known complication of bacterial meningitis but its treatment is uncertain. METHODS AND RESULTS: We report the case of a 53-year-old man with pneumococcal meningitis who developed numerous ischemic lesions in the brainstem and basal ganglia caused by parainfectious vasculitis. Clinical and radiological improvement was observed after delayed corticosteroid initiation. Symptomatic vasculitis relapsed after steroid withdrawal and stabilized after reintroduction of the immunosuppressive therapy. Although the cerebrospinal fluid (CSF) contained high levels of MMP-9 at the time of symptomatic vasculitis, a significant decrease of the enzyme accompanied the introduction of corticotherapy and the regression of vasculitic symptoms. No relation between the level of MMP-9 and the white blood cell count in CSF could be found. CONCLUSION: Parainfectious vasculitis may respond to late corticosteroid treatment. MMP-9 level in CSF may be a marker of vasculitic complication in bacterial meningitis.

Pneumococcal zinc metalloproteinase ZmpC cleaves human matrix metalloproteinase 9 and is a virulence factor in experimental pneumonia.

The ZmpC zinc metalloproteinase of Streptococcus pneumoniae, annotated in the type 4 genome as SP0071, was found to cleave human matrix metalloproteinase 9 (MMP-9). The previously described IgA protease activity was confirmed to be specifically linked to the IgA1-protease/SP1154 zinc metalloproteinase. MMP-9 is a protease cleaving extracellular matrix gelatin and collagen and is activated by proteolytic cleavage like most proteases. MMP-9 is a human protease and is involved in a variety of physiological and pathological matrix degrading processes, including tissue invasion of metastases and opening of the blood-brain barrier. While TIGR4 (serotype 4) and G54 (serotype 19) pneumococcal genome strains have a highly conserved copy of zmpC, the genome of R6 (a derivative of serotype 2 D39 strain) lacks zmpC. Both the analysis for zmpC presence and MMP-9 cleavage activity in various pneumococcal strains showed correlation of ZmpC with MMP-9 cleavage activity. When assaying clinical isolates of S. pneumoniae, the zmpC gene was not found in any of the nasal and conjunctival swab isolates, but it was present in 1 out of 13 meningitis isolates and in 6 out of 11 pneumonia isolates. In a murine pneumonia model, infection with a zmpC-mutant reduced mortality at 3-4 days post-infection by 75%, when compared with infection with wild-type strains. These data indicate that the ZmpC pneumococcal protease may play a role in pneumococcal virulence and pathogenicity in the lung.

Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis.

Bacterial meningitis causes neuronal apoptosis in the hippocampal dentate gyrus, which is associated with learning and memory impairments after cured disease. The execution of the apoptotic program involves pathways that converge on activation of caspase-3, which is required for morphological changes associated with apoptosis. Here, the time course and the role of caspase-3 in neuronal apoptosis was assessed in an infant rat model of pneumococcal meningitis. During clinically asymptotic meningitis (0-12 h after infection), only minor apoptotic damage to the dentate gyrus was observed, while the acute phase (18-24 h) was characterized by a massive increase of apoptotic cells, which peaked at 36 h. In the subacute phase of the disease (36-72 h), the number of apoptotic cells decreased to control levels. Enzymatic caspase-3 activity was significantly increased in hippocampal tissue of infected animals compared to controls at 22 h. The activated enzyme was localized to immature cells of the dentate gyrus, and in vivo activity was evidenced by cleavage of the amyloid-beta precursor protein. Intracisternal administration of the caspase-3-specific inhibitor Ac-DEVD-CHO significantly reduced apoptosis in the hippocampal dentate gyrus. In contrast to a study where the decrease of hippocampal apoptosis after administration of a pan-caspase inhibitor was due to downmodulation of the inflammatory response, our data demonstrate that specific inhibition of caspase-3 did not affect inflammation assessed by TNF-alpha and IL-1beta concentrations in the cerebrospinal fluid space. Taken together, the present results identify caspase-3 as a key effector of neuronal apoptosis in pneumococcal meningitis.

Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway.

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis.

Current concepts in the pathogenesis of meningitis caused by Streptococcus pneumoniae.

In spite of improved antimicrobial therapy, bacterial meningitis still results in brain damage leading to significant long-term neurological sequelae in a substantial number of survivors, as confirmed by several recent studies. Meningitis caused by Streptococcus pneumoniae is associated with a particularly severe outcome. Experimental studies over the past few years have increased our understanding of the molecular mechanisms underlying the events that ultimately lead to brain damage during meningitis. Necrotic damage to the cerebral cortex is at least partly mediated by ischemia and oxygen radicals and therefore offers a promising target for adjunctive therapeutic intervention. Neuronal apoptosis in the hippocampus may represent the major pathological process responsible for cognitive impairment and learning disabilities in survivors. However, the mechanisms involved in causing this damage remain largely unknown. Anti-inflammatory treatment with corticosteroids aggravates hippocampal damage, thus underlining the potential shortcomings of current adjuvant strategies. In contrast, the combined inhibition of matrix metalloproteinase and tumour necrosis factor-alpha converting enzyme protected both the cortex and hippocampus in experimental meningitis, and may represent a promising new approach to adjunctive therapy. It is the hope that a more refined molecular understanding of the pathogenesis of brain damage during bacterial meningitis will lead to new adjunctive therapies.

Meningitis-associated central nervous system complications are mediated by the activation of poly(ADP-ribose) polymerase.

The present study assessed the role of PARP [poly(adenosine diphosphate-ribose) polymerase] activation in experimental pneumococcal meningitis. Mice with a targeted disruption of the PARP 1 gene were protected against meningitis-associated central nervous system complications including blood-brain barrier breaching and increase in intracranial pressure. This beneficial effect was paralleled by a significant reduction in meningeal inflammation, as evidenced by significantly lower cerebrospinal fluid leukocyte counts and interleukin-1beta, -6, and tumor necrosis factor-alpha concentrations in the brain (compared with infected wild-type mice). The reduction in inflammation and central nervous system complications was associated with an improved clinical status of infected, PARP 1-deficient mice. A similar protective effect was achieved by PARP inhibition using 3-aminobenzamide, the pharmacologic efficacy of which was confirmed by a marked attenuation of meningitis-induced poly(ADP)ribose formation. When the rat brain-derived endothelial cell line GP8.3 was cocultured with macrophages, exposure to pneumococci induced endothelial cell death and was paralleled by PARP activation and a reduction in the oxidized form of cellular nicotinamide adenine dinucleotide content. Treatment with 3-aminobenzamide significantly attenuated cellular nicotinamide adenine dinucleotide depletion and pneumococci-induced cytotoxicity. Thus, PARP activation seems to play a crucial role in the development of meningitis-associated central nervous system complications and pneumococci-induced endothelial injury. Inhibitors of PARP activation could provide a potential therapy of acute bacterial meningitis.

Reactive nitrogen species contribute to blood-labyrinth barrier disruption in suppurative labyrinthitis complicating experimental pneumococcal meningitis in the rat.

Sensorineural hearing damage is a frequent complication of bacterial meningitis, affecting as many as 30% of survivors of pneumococcal meningitis. There is a substantial body of evidence that oxidants, such as reactive nitrogen species (RNS), are central mediators of brain damage in experimental bacterial meningitis. In the present study, we investigated whether RNS also contribute to the pathophysiology of suppurative labyrinthitis in our well-established rat model of pneumococcal meningitis. In all infected rats, but not in uninfected controls, we observed suppurative labyrinthitis. Cochlear inflammation was accompanied by severe blood-labyrinth barrier (BLB) disruption as evidenced by increased Evans Blue extravasation. Furthermore, increased cochlear expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry. Colocalization of iNOS and tyrosine nitration (a marker of RNS attack) indicated that nitric oxide (NO) produced by iNOS contributes to oxidative cochlear damage through the action of RNS. To determine the pathophysiological role of RNS in BLB disruption, rats were treated with peroxynitrite scavengers (MnTBAP and uric acid, UA). Six h after adjunctive treatment with 300 mg/kg i.p. UA or 15 mg/kg i.p. MnTBAP+100 mg/kg i.p. ceftriaxone, BLB disruption was significantly reduced compared with that in infected animals treated only with ceftriaxone. Therefore, we conclude that RNS are involved in the breaching of the BLB during meningogenic pneumococcal labyrinthitis.

Differential expression of nitric oxide synthases in bacterial meningitis: role of the inducible isoform for blood-brain barrier breakdown.

The aim of the study was to determine the differential expression of nitric oxide (NO) synthase (NOS) isoforms and the pathophysiologic relevance of inducible NOS (iNOS) in experimental pneumococcal meningitis. By use of reverse transcription-polymerase chain reaction analysis, immunohistochemistry, and Western blotting, increased brain mRNA and increased protein levels of endothelial NOS (eNOS) and iNOS were detected 24 h after intracisternal pneumococcal inoculation. In iNOS-deficient mice, disruption of the blood-brain barrier (BBB) was significantly reduced, compared with that in wild-type mice. This beneficial effect of iNOS deficiency was associated with a lack of nitrotyrosine immunoreactivity. Furthermore, brain protein levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha and brain mRNA levels of macrophage inflammatory protein (MIP)-1alpha and MIP-2 were significantly reduced in infected animals lacking iNOS. These findings suggest that (1) not only iNOS but also eNOS is up-regulated in the acute phase of experimental bacterial meningitis, and (2) iNOS-derived NO contributes to peroxynitrite formation and BBB breaching in this disease.