A method to isolate and cryopreserve cerebrospinal fluid mononuclear cells from external ventricular drains to investigate immunological processes in acute brain injuries.

The inflammatory response to acute brain injuries is a key contributor to subsequent outcome. The study of local central nervous system inflammatory responses is hindered by raised intracranial pressure precluding cerebrospinal fluid sampling by lumbar puncture. External ventricular drains are sited in some acute brain injury patients to divert cerebrospinal fluid and thus reduce intracranial pressure, and represent a potential route to safely gather large volumes of cerebrospinal fluid for immunological studies. In this manuscript we show that mononuclear cells can be isolated from cerebrospinal fluid collected from external ventricular drains, and that the large volumes of cerebrospinal fluid available yield sufficient mononuclear cells to allow cryopreservation. Prolonged storage of cerebrospinal fluid in the external ventricular drain collection bag can alter the phenotype of cells recovered, but the predicted effect of this can be estimated for a given flow cytometry panel by assessing the changes in peripheral blood mononuclear cells exposed to the same conditions. The described method will allow clinical studies of acute brain injuries to investigate the immunological processes occurring within the central nervous system compartment, rather than relying on changes in the peripheral circulation.

Cryptococcus neoformans ex vivo capsule size is associated with intracranial pressure and host immune response in HIV-associated cryptococcal meningitis.

BACKGROUND: The Cryptococcus neoformans polysaccharide capsule is a well-characterized virulence factor with immunomodulatory properties. The organism and/or shed capsule is postulated to raise intracranial pressure (ICP) in cryptococcal meningitis (CM) by mechanical obstruction of cerebrospinal fluid (CSF) outflow. Little is known regarding capsule phenotype in human cryptococcosis. We investigated the relationship of ex vivo CSF capsular phenotype with ICP and CSF immune response, as well as in vitro phenotype. METHODS: In total, 134 human immunodeficiency virus (HIV)-infected Ugandan adults with CM had serial lumbar punctures with measurement of CSF opening pressures, quantitative cultures, ex vivo capsule size and shedding, viscosity, and CSF cytokines; 108 had complete data. Induced capsular size and shedding were measured in vitro for 48 C. neoformans isolates. RESULTS: Cryptococcal strains producing larger ex vivo capsules in the baseline (pretreatment) CSF correlated with higher ICP (P = .02), slower rate of fungal clearance (P = .02), and paucity of CSF inflammation, including decreased CSF white blood cell (WBC) count (P < .001), interleukin (IL)-4 (P = .02), IL-6 (P = .01), IL-7 (P = .04), IL-8 (P = .03), and interferon γ (P = .03). CSF capsule shedding did not correlate with ICP. On multivariable analysis, capsule size remained independently associated with ICP. Ex vivo capsular size and shedding did not correlate with that of the same isolates grown in vitro. CONCLUSIONS: Cryptococcal capsule size ex vivo is an important contributor to virulence in human cryptococcal meningitis.

The effect of ubiquitin on immune response after controlled cortical impact injury.

BACKGROUND: Previously, we reported that exogenous ubiquitin reduces cortical contusion volume and tends to reduce brain water content after controlled cortical impact injury Controlled Cortical Impact Injury (CCII) in rats. The mechanisms how exogenous ubiquitin exerts these effects remain unclear. Some studies revealed ubiquitin's immune modulatory abilities; therefore, we hypothesized that ubiquitin influences the local innate inflammatory response after CCII. METHODS: Sprague-Dawley rats were exposed to CCII and randomized to either 1.5 mg/kg ubiquitin or 0.9% NaCl intravenously within 5 minutes after CCII. Immune cells were immunohistochemically stained with OX-42, myeloperoxidase (MPO), HIS48, ED1, and glial fibrillary acidic protein (GFAP). Apoptosis was analyzed by using terminal desoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). Levels of interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1 receptor antagonist (IL-1ra) were quantified using real-time reverse-transcriptase polymerase chain reaction (rt-PCR). RESULTS: ED1-positive cells were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7 (823±182 cells/mm² vs. 550±246 cells/mm²; p=0.04). IL-10 expression after 3 days was significantly lower in the verum group (1.065¹°â»5±0.6093¹°â»5 vs. 2.266¹°â»5±1.244¹°â»5 relative messenger RNA expression; p=0.04) and TNF-α-levels tended to be higher in the verum group (22.01¹°â»5±10.87¹°â»5 vs. 9.34¹°â»5±4.44¹°â»5 relative messenger RNA; p=0.096). Quantification of apoptotic cells did not differ between the groups. CONCLUSION: Exogenous ubiquitin modulates the immune response by influencing the infiltration of macrophages or activated microglia and the expression of IL-10 and possibly TNF-α after CCII. The effects of these changes in immune response on posttraumatic neurodegeneration still need to be clarified.

Increased intracranial pressure induces a rapid systemic interleukin-10 release through activation of the sympathetic nervous system.

There is a bi-directional communication between the immune and central nervous system. In this context, it is known that patients with traumatic brain injury suffered from systemic immunodepression and an increased risk to develop infectious complications. We investigated the role of an increased intracranial pressure (ICP) and sympathetic activation on systemic immune changes. A sustained increase in ICP was achieved by inflation of a subdural balloon. At different time points, plasma levels of the anti-inflammatory cytokine, interleukin (IL)-10, were measured. Furthermore, the effect of a sympathetic blockade by co-administration of the beta2-adreoreceptor antagonist, propranolol, was evaluated. Finally, we examined the impact of epinephrine infusion on blood IL-10 levels. We showed that an increase in ICP with activation of the sympathetic nervous system was able to induce systemic release of IL-10. This effect was blocked by administration of the beta2-adreoreceptor antagonist. Furthermore, epinephrine infusion directly induced systemic release of IL-10. Our data suggested that sympathetic activation with release of epinephrine may induce systemic immunodepression with risk of infectious complications in brain-injured patients.

Toll-like receptor 2 participates in mediation of immune response in experimental pneumococcal meningitis.

Heterologous expression of Toll-like receptor (TLR)2 and CD14 in Chinese hamster ovary fibroblasts was reported to confer responsiveness to pneumococcal peptidoglycan. The present study characterized the role of TLR2 in the host immune response and clinical course of pneumococcal meningitis. Pneumococcal infection of mice caused a significant increase in brain TLR2 mRNA expression at both 4 and 24 h postchallenge. Mice with a targeted disruption of the TLR2 gene (TLR2-/-) showed a moderate increase in disease severity, as evidenced by an aggravation of meningitis-induced intracranial complications, a more pronounced reduction in body weight and temperature, and a deterioration of motor impairment. These symptoms were associated with significantly higher cerebellar and blood bacterial titers. Brain expression of the complement inhibitor complement receptor-related protein y was significantly higher in infected TLR2-/- than in wild-type mice, while the expression of the meningitis-relevant inflammatory mediators IL-1beta, TNF-alpha, IL-6, macrophage-inflammatory protein (MIP)-2, inducible NO synthase, and C3 was similar in both genotypes. We first ectopically expressed single candidate receptors in HEK293 cells and then applied peritoneal macrophages from mice lacking TLR2 and/or functional TLR4 for further analysis. Overexpression of TLR2 and TLR4/MD-2 conferred activation of NF-kappaB in response to pneumococcal exposure. However, pneumococci-induced TNF-alpha release from peritoneal macrophages of wild-type and TLR2/functional TLR4/double-deficient mice did not differ. Thus, while TLR2 plays a significant role in vivo, yet undefined pattern recognition receptors contribute to the recognition of and initiation of the host immune defense toward Streptococcus pneumoniae infection.

Detectable concentrations of Fas ligand in cerebrospinal fluid after severe head injury.

When the cell surface molecule Fas is triggered by its agonist Fas ligand the result is apoptosis of these cells and tissue destruction. To elucidate the pathophysiological relevance of Fas ligand in patients with cerebral oedema caused by trauma, we examined its concentrations in cerebrospinal fluid in 18 patients using specific ELISA. Serum and cerebrospinal fluid from healthy people and injured patients without head trauma did not contain detectable Fas ligand. In contrast, cerebrospinal fluid from patients with severe brain injury contained high concentrations of Fas ligand without detectable concentrations in serum. Soluble Fas ligand concentrations in cerebrospinal fluid correlated significantly with severity of brain injury. The Fas-Fas ligand-system may have a pivotal role in causing oedema and local tissue destruction in the brain after severe head injury.