Manganese exposure in juvenile C57BL/6 mice increases glial inflammatory responses in the substantia nigra following infection with H1N1 influenza virus.

Infection with Influenza A virus can lead to the development of encephalitis and subsequent neurological deficits ranging from headaches to neurodegeneration. Post-encephalitic parkinsonism has been reported in surviving patients of H1N1 infections, but not all cases of encephalitic H1N1 infection present with these neurological symptoms, suggesting that interactions with an environmental neurotoxin could promote more severe neurological damage. The heavy metal, manganese (Mn), is a potential interacting factor with H1N1 because excessive exposure early in life can induce long-lasting effects on neurological function through inflammatory activation of glial cells. In the current study, we used a two-hit model of neurotoxin-pathogen exposure to examine whether exposure to Mn during juvenile development would induce a more severe neuropathological response following infection with H1N1 in adulthood. To test this hypothesis, C57BL/6 mice were exposed to MnCl2 in drinking water (50 mg/kg/day) for 30 days from days 21-51 postnatal, then infected intranasally with H1N1 three weeks later. Analyses of dopaminergic neurons, microglia and astrocytes in basal ganglia indicated that although there was no significant loss of dopaminergic neurons within the substantia nigra pars compacta, there was more pronounced activation of microglia and astrocytes in animals sequentially exposed to Mn and H1N1, as well as altered patterns of histone acetylation. Whole transcriptome Next Generation Sequencing (RNASeq) analysis was performed on the substantia nigra and revealed unique patterns of gene expression in the dual-exposed group, including genes involved in antioxidant activation, mitophagy and neurodegeneration. Taken together, these results suggest that exposure to elevated levels of Mn during juvenile development could sensitize glial cells to more severe neuro-immune responses to influenza infection later in life through persistent epigenetic changes.

Parechovirus and enterovirus infections in neonates.

BACKGROUND: Parechovirus and enterovirus belong to a family of Picornaviridae, non- enveloped, small-sized RNA viruses, responsible for multiple human diseases. Recent introduction of molecular tests enabled the identi cation of parechovirus and enterovirus infections. Our aim was a retrospective analysis of signs and symptoms associated with confirmed parechovirus or enterovirus infections among children treated in the Department of Neonatology, St. Louis Regional Children's Hospital in Kraków, Poland. METHODS: Based on laboratory records, we identified all cases of parecho- or enterovirus infections confirmed by identification of viral RNA in nasal swab or cerebrospinal fluid samples. Hospital records and laboratory tests results of selected patients were then analyzed, and selected data were summarized, with emphasis on clinical and laboratory findings at admission. RESULTS: We identified 11 cases of parechovirus and three of enterovirus infections. All cases were neonates admitted to hospital with fever and irritability. Except for leukopenia in 50% of patients, no significant abnormalities were noted in blood counts and serum biochemistry, including low C-reactive protein and procalcitonin. In nine cases, cerebrospinal fluid was collected, the fluid protein concentrations and cell counts were moderately increased. Final diagnosis was meningitis in 12 children, and other viral infections in two. CONCLUSIONS: Viral infection, including parecho- and enteroviruses, should be considered in the etiology of fever and meningitis in neonates. The available molecular tests allow for detection of viral genetic material even in a scant biological specimen collected from neonates.

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)-carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

Application value of procalcitonin in patients with central nervous system infection.

OBJECTIVE: To study the application value of procalcitonin (PCT) in patients with central nervous system (CNS) infection. PATIENTS AND METHODS: A total of 66 patients, including 24 patients with suppurative meningitis, 20 patients with viral meningitis and 22 patients with tuberculous meningitis, were enrolled. 20 patients admitted to the hospital due to epilepsy or headache without infection in the same period were enrolled as the control group. PCT, high-sensitivity C-reactive protein (Hs-CRP), high-sensitivity C-reactive protein (Hs-CRP), protein quantification, chloride and glucose in serum and cerebrospinal fluid, were collected. RESULTS: The serum PCT level in suppurative meningitis group was significantly higher than that in other three groups. The dynamic monitoring of suppurative meningitis group on admission, at 72 h and 1 week after treatment showed that the serum PCT level was significantly decreased. PCT levels in cerebrospinal fluid in suppurative meningitis group, viral meningitis group and tuberculous meningitis group were decreased successively, and the differences were statistically significant. The detection of PCT in cerebrospinal fluid was more valuable than serum PCT detection in distinguishing tuberculous meningitis from viral meningitis. Continuous monitoring of changes in PCT in cerebrospinal fluid showed that there was no statistically significant difference before and after treatment. PCT level in cerebrospinal fluid was positively correlated with the serum PCT, cerebrospinal fluid white blood cell (WBC), and protein content in cerebrospinal fluid. CONCLUSIONS: The dynamic changes of serum PCT in patients with suppurative meningitis can be used to evaluate the disease, guide the clinical medication, and monitor the prognosis.

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

PURPOSE: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). METHODS: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. RESULTS: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CONCLUSIONS: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation.

Meningitis víricas en adultos en un hospital comarcal / Viral meningitis in adults in a district hospital

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The diagnostic value of cytokine and nitric oxide concentrations in cerebrospinal fluid for the differential diagnosis of meningitis.

PURPOSE: In several cases of meningitis routinely used diagnostic procedures are unable to identify the cause of this disease. The objective of the present study was to determine whether proinflammatory cytokine (tumour necrosis factor (TNF-α), interleukin-1ß (IL-1ß), interleukin-8 (IL-8)) and nitric oxide (NO) concentrations in the CSF are useful markers for the differential diagnosis of meningitis. MATERIAL AND METHODS: Sixty-seven patients (42 patients with bacterial meningitis and 25 patients with viral meningitis) were included in the present study. In the investigated group, the TNF-α, IL-1ß and IL-8 concentrations in the CSF samples collected on the day of admission were assessed. Furthermore, the NO concentrations were assessed in 23 patients. RESULTS: The results revealed that the measurement of proinflammatory cytokines in CSF can aid in a differential diagnosis. In particular, a high concentration of TNF-α may be a sensitive and specific marker of a bacterial aetiology of the neuroinfection. In the present study, TNF-α concentrations greater than 75.8 pg/ml differentiated between bacterial and viral meningitis with 100% sensitivity and specificity. The NO concentration in the CSF was also significantly greater in patients with bacterial meningitis than in those with viral meningitis. CONCLUSIONS: The assessment of TNF-α, IL-1ß and IL-8 concentrations in the CSF is useful in the differential diagnosis of neuroinfection. Because many factors may influence NO production in the central nervous system (CNS), it is not clear whether NO values can be used for the differential diagnosis of meningitis, and further studies are required.

Dengue virus 3 clinical isolates show different patterns of virulence in experimental mice infection.

Dengue virus (DENV) may cause symptomatic infection with mild, undifferentiated febrile illness called classical dengue fever (DF) or a more severe disease, potentially fatal, known as dengue hemorrhagic fever (DHF) or dengue shock syndrome. The pathogenesis of DHF is based on the virulence of the infecting DENV and depends on the infecting serotypes and genotypes; it is also based on the immunopathogenesis that is mediated by host immune responses, including dengue virus-cross-reactive antibodies that augment the severity of infections. Involvement of central nervous system (CNS) is extensively described. The present study describes the virulence of DENV-3 isolates in a mouse model by intracranial (i.c.) inoculation with genotypes I and III. Our data suggest that, in this experimental model, DENV-3 genotype I may have the propensity to cause neurological disease in mice, whereas the genotype III is associated with asymptomatic infection in mice. Additionally, the symptomatic mice show a decrease of white blood cell count, infectious DENV in the brains and alterations in levels of IFN-gamma, IL-6 and MCP-1. The results confirm the mouse model as a way to study the biology of DENV-3 isolates and to improve the knowledge about the neurovirulence of the different genotypes of DENV.

Regulation of activin A synthesis in microglial cells: pathophysiological implications for bacterial meningitis.

Previous studies have shown that activin A, a neuroprotective cytokine and dimeric polypeptide composed of two betaA subunits, is elevated in the cerebrospinal fluid of patients suffering from bacterial meningitis. In this study, to elucidate further the functional significance and pathophysiological implications of these findings, we demonstrated that microglial cells are not only the source but also the target cells of activin A in the central nervous system: immunohistochemistry and RT-PCR revealed expression of activin subunit betaA mRNA as well as activin receptor type I and type II mRNA in rat microglia in vitro. Further studies showed that activin enhances microglial proliferation and decreases the gamma-interferon-induced synthesis of nitric oxide, one of several microglial mediators involved in the inflammatory response in microglia activation. Furthermore, quantitative RT-PCR, Western blotting, and ELISA showed an inhibitory effect of activin on inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta gene and protein levels after lipopolysaccharide treatment. We suggest that the increased synthesis of activin A is directly involved, via influence on microglia cell functions, in the modulation of the inflammatory response in bacterial meningitis.

[Analysis of 62 adult patients with viral meningitis].

OBJECTIVE: To investigate the clinical manifestation, cerebrospinal fluid (CSF) and auxiliary examination findings of adult viral meningitis. METHODS: 62 adult patients with viral meningitis were retrospectively analyzed. RESULTS: Headache occurred in all the 62 (100%) patients, fever occurred in 61 (98%) patients, meningeal irritation sign occurred in 48 (77%) patients. The abduction of left eye was limited in one patient. Seizure occurred in 2 patients. The mean duration time was 17 days, 93% patients less than 30 days. The pressure of CSF increased in 80% patients, leukocyte counts increased in 91% patients, protein level increased in 81% patients, chloride level was normal in 35% patients and slightly lower in 65% patients, glucose level was normal in 94% patients. 7 patients had positive IgM antibody of Coxackievirus B group both in serum and CSF, one patient had positive IgM antibody of EB virus in CSF. Cranial CT scan had no special findings in all patients. 23 patients performed MRI examination, meningeal enhancement occurred in 9 patients. 52% patients had abnormal EEG, mainly increased local or diffuse slow waves. CONCLUSION: Adult viral meningitis was a kind of self-limited disease, chloride level was slightly lower in more than half patients, meningeal enhancement was detected in MRI in part patients.

Hepatitis B en niños y gestantes / Hepatitis B in children and pregnant women

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Fatal Epstein-Barr virus meningitis: an autopsy report.

A 3-year-old Asian female presented with fever for 1 week and neck swelling for 1 day. Serology revealed a recent Epstein-Barr virus (EBV) infection. Late on the evening of admission, she developed confusion and would not follow commands. A CT scan showed diffuse cerebral edema and a cerebral flow scan demonstrated no blood flow to the brain. She was declared brain dead and expired on the following day. At autopsy, the brain weighted 1175 grams and grossly showed significant edema. Microscopically, the entire neuraxis revealed extensive leptomeningeal infiltrate of mainly CD8+ T lymphocytes, the majority of which expressed activated markers, HLA-DR and/or CD45RO, and monocytes/macrophages with intermixed numerous apoptotic/karyorrhectic nuclear fragments. These nuclear fragments were considered to be due to apoptosis of the expanded population of CD8+ T lymphocytes. Focal venulitis was noted. EBV-encoded small nuclear RNA in situ hybridization revealed positivity in the occasional lymphocytes. Interestingly, most intraparenchymal as well as leptomeningeal vascular endothelium showed HLA-DR immunoreactivity. This finding has been reported primarily in the acute inflammatory/demyelinating conditions, not in the viral meningitis/meningoencephalitis, and was thought to be related to cytokines due to widespread inflammation in our case. Massive edema secondary to severe EBV-meningitis can be fatal.

CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system.

IFN-gamma-inducible protein 10/CXCL10 is a chemokine associated with type 1 T cell responses, regulating the migration of activated T cells through binding to the CXCR3 receptor. Expression of both CXCL10 and CXCR3 are observed during immunopathological diseases of the CNS, and this receptor/ligand pair is thought to play a central role in regulating T cell-mediated inflammation in this organ site. In this report, we investigated the role of CXCL10 in regulating CD8(+) T cell-mediated inflammation in the virus-infected brain. This was done through analysis of CXCL10-deficient mice infected intracerebrally with lymphocytic choriomeningitis virus, which in normal immunocompetent mice induces a fatal CD8(+) T cell-mediated meningoencephalitis. We found that a normal antiviral CD8(+) T cell response was generated in CXCL10-deficient mice, and that lack of CXCL10 had no influence on the accumulation of mononuclear cells in the cerebrospinal fluid. However, analysis of the susceptibility of CXCL10-deficient mice to lymphocytic choriomeningitis virus-induced meningitis revealed that these mice just like CXCR3-deficient mice were partially resistant to this disease, whereas wild-type mice invariably died. Furthermore, despite marked up-regulation of the two remaining CXCR3 ligands: CXCL9 and 11, we found a reduced accumulation of CD8(+) T cells in the brain parenchyma around the time point when wild-type mice succumb as a result of CD8(+) T cell-mediated inflammation. Thus, taken together these results indicate a central role for CXCL10 in regulating the accumulation of effector T cells at sites of CNS inflammation, with no apparent compensatory effect of other CXCR3 ligands.

Serum ferritin levels in West Nile encephalitis.

West Nile encephalitis (WNE) presents clinically as aseptic meningitis, meningoencephalitis, encephalitis, or acute flaccid paralysis. Non-specific laboratory findings, e.g., leukopenia and thrombocytopenia, accompany WNE. Lymphopenia is marked and prolonged with WNE. Three patients with WNE were found to have elevated serum ferritin levels. Severity seemed to be directly related to serum ferritin levels. Although preliminary, the results suggested that serum ferritin levels >or= 500 ng/mL (normal range 5-187 ng/mL) occur late with WNE, and not in a control group of patients with viral meningitis or encephalitis.

Identification of a matrix-degrading phenotype in human tuberculosis in vitro and in vivo.

Tuberculous meningitis is characterized by cerebral tissue destruction. Monocytes, pivotal in immune responses to Mycobacterium tuberculosis, secrete matrix metalloproteinase-9 (MMP-9), which facilitates leukocyte migration across the blood-brain barrier, but may cause cerebral injury. In vitro, human monocytic (THP-1) cells infected by live, virulent M. tuberculosis secreted MMP-9 in a dose-dependent manner. At 24 h, MMP-9 concentrations increased 10-fold to 239 +/- 75 ng/ml (p = 0.001 vs controls). MMP-9 mRNA became detectable at 24--48 h. In contrast, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) gene expression and secretion were similar to constitutive levels from controls at 24 h and increased just 5-fold by 48 h. In vivo investigation revealed MMP-9 concentration per leukocyte in cerebrospinal fluid (CSF) from tuberculous meningitis patients (n = 23; median (range), 3.19 (0.19--31.00) ng/ml/cell) to be higher than that in bacterial (n = 12; 0.23 (0.01--18.37) ng/ml/cell) or viral meningitis (n = 20; 0.20 (0.04--31.00) ng/ml/cell; p < 0.01). TIMP-1, which was constitutively secreted into CSF, was not elevated in tuberculous compared with bacterial meningitis or controls. Thus, a phenotype in which MMP-9 activity is relatively unrestricted by TIMP-1 developed both in vitro and in vivo. This is functionally significant, since MMP-9 concentrations per CSF leukocyte (but not TIMP-1 concentrations) were elevated in fatal tuberculous meningitis and in patients with signs of cerebral tissue damage (unconsciousness, confusion, or neurological deficit; p < 0.05). However, MMP-9 activity was unrelated to the severity of systemic illness. In summary, M. tuberculosis-infected monocytic cells develop a matrix-degrading phenotype, which was observed in vivo and relates to clinical signs reflecting cerebral injury in tuberculous meningitis.

Direct and mononuclear cell mediated effects on interleukin 6 production by glioma cells in infection with herpes simplex virus type 1.

To clarify the mechanism of interleukin (IL)-6 elevation in the cerebrospinal fluid of viral meningitis and/or encephalitis patients, we investigated how herpes simplex virus type 1 (HSV1)-infection enhances IL-6 production in human glioma cells (the U373MG and T98G cells). Although human glioma cells did not show enhanced IL-6 production by direct HSV1-infection, the cell-free supernatant from HSV1-stimulated mononuclear cells (MNC) culture and lipopolysaccharide, as a positive control, markedly elevated IL-6 production at both mRNA and polypeptide levels. Ultra violet-irradiated HSV1 induced the secretion of the IL-6 inducing factor(s) from MNC, whereas heat-inactivated HSV1 did not show this activity. This finding indicated that the adsorption of virus on the surface of MNC may be sufficient for induction of secretion. The supernatant from the culture of HSV1-stimulated MNC contained detectable amounts of IL-1beta, tumor necrosis factor (TNF) alpha, interferon (IFN) gamma and IL-6, and its IL-6-inducing activity was inhibited only by anti-IL-1beta antibodies. Moreover, recombinant IL-1beta markedly enhanced IL-6 production in glioma cells with a concomitant elevation of its mRNA level. Taken together, the results suggest that in HSV1-infection of the CNS, enhancement of IL-6 production in glial cells is mediated not by direct infection to glial cells but rather by IL-1beta released from HSV1-stimulated MNC. These findings may help elucidate the mechanisms underlying cerebro-parenchymal inflammatory progression and repair in herpes simplex encephalitis.

Matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF): elevated levels are primarily related to CSF cell count.

Matrix metalloproteinase-9 (MMP-9) was investigated by enzyme-linked immunosorbent assay (ELISA) and zymography in 111 paired CSF and serum samples from patients with various neurological disorders. In 20 patients with blood-brain barrier (BBB) impairment but normal CSF cell count, elevated levels of MMP-9 were not observed by ELISA measurement. Another 11 patients characterized in the same way, exhibited only slightly increased MMP-9 levels. In contrast, in 12 patients with intact BBB but elevated CSF cell count, MMP-9 was increased too. It was shown by the more sensitive zymography that MMP-9 increased if CSF cell count exceeded five cells per microl. Spearman rank statistics revealed that MMP-9 concentration in CSF correlated with CSF cell count (r=0.755; P<0.0001), but not with CSF/serum albumin ratio (Q(Alb)) (r=0.212; P=0.057), a measure for BBB impairment. Moreover, the CSF/serum MMP-9 ratio (Q(MMP-9)) did not correlate with Q(Alb)(r=0.192; P=0.100). By use of a Boyden chamber, in which granulocytes migrated through a reconstituted basement membrane, it was demonstrated that the MMP-9 concentration in the lower chamber correlated very significantly with the number of accumulated cells (r(2)=0.7692; P<0.0001). The meaning of the increase of MMP-9 in CSF is critically discussed.

Clinical utility of the polymerase chain reaction for diagnosis of enteroviral meningitis in infancy.

OBJECTIVE: To determine the utility of polymerase chain reaction (PCR) assay of cerebrospinal fluid (CSF), serum, and urine for rapid diagnosis of enteroviral meningitis in infants 3 months of age and younger. STUDY DESIGN: We identified prospectively infants 3 months of age and younger coming to the emergency department with fever whose examination included a lumbar puncture, blood culture, or both. Samples of CSF, serum, urine, throat, and stool specimens were collected for viral culture and, with the exception of stool, for PCR assay. Those infants who had not received prior antibiotic therapy and had sterile bacterial cultures of CSF, blood, and urine were selected for the present analysis. RESULTS: A total of 259 specimens for viral culture and 203 specimens for PCR assay were collected from 64 infants. Comparison of results of PCR assay of CSF with viral culture, the gold standard for diagnosis of enteroviral meningitis, demonstrated a sensitivity of 100% and a specificity of 90%. Because enteroviruses are not always detectable by culture, the following modified standard was established to define enteroviral meningitis: either CSF pleocytosis, sterile bacterial cultures and detection of an enterovirus in stool culture or positive viral culture of CSF, or both. With this modified definition, the sensitivity and specificity of the PCR assay of CSF were 92% and 94%, respectively. PCR assay of serum and urine offered no benefit over PCR assay of CSF alone for diagnosis of meningitis. CONCLUSION: PCR assay of CSF is useful for the rapid and reliable diagnosis of enteroviral meningitis. Application of this technique in the clinical setting can potentially diminish unnecessary hospitalization and use of antibiotics.

Cerebrospinal fluid hypoxanthine, xanthine and uric acid levels may reflect glutamate-mediated excitotoxicity in different neurological diseases.

Glutamate-mediated excitotoxicity is associated with adenosine triphosphate (ATP) degradation and generation of oxygen radicals. Hypoxanthine and lactate depict energetic impairment, while xanthine and uric acid reflect activity of radical producing xanthine oxidase. Cerebrospinal fluid (CSF) glutamate, hypoxanthine, lactate, xanthine, and uric acid were investigated in neurological patients. In multiple sclerosis, myelopathy, stroke, epilepsy and viral meningitis glutamate, hypoxanthine, xanthine, and uric acid are increased 2-3-fold compared to controls. Lactate is only elevated in meningitis. Normal lactate dehydrogenase (LDH) levels and absent correlation between the albumin ratio and neurochemical parameters exclude an artificial increase due to cell lysis and barrier damage. Absent correlation between neurochemical parameters within each patient group is most likely related to preserved glial and neuronal uptake mechanisms. CSF hypoxanthine, xanthine, and uric acid levels appear superior to lactate in reflecting glutamate-mediated excitotoxicity in neurological patients.