Expression and functional relevance of UGT1A4 in a cohort of human drug-resistant epileptic brains.

PURPOSE: Brain drug bioavailability is regulated by the blood-brain barrier (BBB). It was recently suggested that cytochrome P450 (CYP) enzymes could act in concert with multidrug transporter proteins to regulate drug penetration and distribution into the diseased brain. The possibility that phase II metabolic enzymes could be expressed in the epileptic brain has been not evaluated. Phase II enzymes are involved in the metabolism of common antiepileptic drugs (AEDs). METHODS: Phase II enzyme UGT1A4 brain expression was evaluated in temporal lobe resections from patients with epilepsy. UGT1A4 expression was determined by western blot and immunocytochemistry in primary cultures of human drug-resistant brain endothelial human brain epileptic endothelial cells (EPI-EC)s and commercially available control cells human brain microvascular endothelial cells (HBMECs). Lack of DNA condensation measured by 4',6-diamidino-2-phenylindole (DAPI) was used as a surrogate marker of cell viability and was correlated to UGT1A4 expression high performance liquid chromatography ultraviolet detection (HPLC-UV) was used to quantify lamotrigine metabolism by EPI-EC and HBMEC. The appearance of the specific lamotrigine metabolite, 2-n glucuronide (MET-1), was also evaluated. Lamotrigine and MET-1 levels were measured in selected surgical brain and matched blood samples. KEY FINDINGS: UGT1A4 expression was observed in BBB endothelial cells and neurons. Our quantification study revealed variable levels of UGT1A4 expression across the brain specimens analyzed. Neurons devoid of UGT1A4 expression displayed nuclear DAPI condensation, a sign of cellular distress. UGT1A4 overexpression in EPI-EC, as compared to HBMEC, was reflected by a proportional increase in lamotrigine metabolism. The lamotrigine metabolite, MET-1, was formed in vitro by EPI-EC and, to a lesser extent, by HBMEC. HPLC-UV measurements of brain and blood samples obtained from patients receiving lamotrigine prior to surgery revealed the presence of lamotrigine and its metabolites in the brain. SIGNIFICANCE: These initial results suggest the presence of a phase II enzyme in the epileptic brain. Further studies are required to fully describe the pattern of brain UGT1A4 expression in relation to clinical variables and drug resistance.

The role of shear stress in Blood-Brain Barrier endothelial physiology.

BACKGROUND: One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain barrier (BBB) phenotype is shear stress (SS). With the use of a well established humanized dynamic in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions. RESULTS: Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER) and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc.) was also upregulated.RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase) were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases) were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample) of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased the expression levels of tight junction proteins. In addition, regulatory enzymes of the Krebb's cycle (aerobic glucose metabolism) were also upregulated. Furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for SS. CONCLUSIONS: Genomic and proteomic analyses are currently used to examine BBB function in healthy and diseased brain and characterize this dynamic interface. In this study we showed that SS plays a key role in promoting the differentiation of vascular endothelial cells into a truly BBB phenotype. SS affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. BBB dysfunction has been observed in many neurological diseases, but the causes are generally unknown. Our study provides essential insights to understand the role played by SS in the BBB formation and maintenance.

Cellular localization and functional significance of CYP3A4 in the human epileptic brain.

PURPOSE: Compelling evidence supports the presence of P450 enzymes (CYPs) in the central nervous system (CNS). However, little information is available on the localization and function of CYPs in the drug-resistant epileptic brain. We have evaluated the pattern of expression of the specific enzyme CYP3A4 and studied its co-localization with MDR1. We also determined whether an association exists between CYP3A4 expression and cell survival. METHODS: Brain specimens were obtained from eight patients undergoing resection to relieve drug-resistant seizures or to remove a cavernous angioma. Each specimen was partitioned for either immunostaining or primary culture of human endothelial cells and astrocytes. Immunostaining was performed using anti-CYP3A4, MDR1, GFAP, or NeuN antibodies. High performance liquid chromatography-ultraviolet (HPLC-UV) analysis was used to quantify carbamazepine (CBZ) metabolism by these cells. CYP3A4 expression was correlated to DAPI) condensation, a marker of cell viability. Human embryonic kidney (HEK) cells were transfected with 4',6-diamidino-2-phenylindole (CYP3A4 to further evaluate the link between CYP3A4 levels, CBZ metabolism, and cell viability. KEY FINDINGS: CYP3A4 was expressed by blood-brain barrier (BBB) endothelial cells and by the majority of neurons (75 ± 10%). Fluorescent immunostaining showed coexpression of CYP3A4 and MDR1 in endothelial cells and neurons. CYP3A4 expression inversely correlated with DAPI nuclear condensation. CYP3A4 overexpression in HEK cells conferred resistance to cytotoxic levels of carbamazepine. CYP3A4 levels positively correlated with the amount of CBZ metabolized. SIGNIFICANCE: CYP3A4 brain expression is not only associated with drug metabolism but may also represent a cytoprotective mechanism. Coexpression of CYP3A4 and MDR1 may be involved in cell survival in the diseased brain.

Modulation of peripheral cytotoxic cells and ictogenesis in a model of seizures.

PURPOSE: A link between seizure susceptibility, blood-brain barrier (BBB) failure, and the activation of peripheral white blood cells has been recently proposed. However, the molecular players involved in this cascade of events are unknown. We tested the hypothesis that immunosupression by splenectomy or lack of perforin, a downstream factor of natural killer (NK) and cytotoxic T cells, could reduce seizure onset. METHODS: Pilocarpine was used to induce seizures in adult rats wild-type and perforin-deficient mice. Splenectomy was performed prior to pilocarpine injection. Seizure onset was evaluated by electroencephalography (EEG) and joint time-frequency analysis. Spleens from control and pilocarpine-treated groups were analyzed for anatomical changes and CD3+ cell content. BBB damage was assessed by measuring albumin parenchymal extravasation. Fluorescence-activated cell sorting (FACS) analysis was performed on spleen and brain tissue of wild-type and perforin-deficient mice treated, or not, with pilocarpine. KEY FINDINGS: Splenectomy significantly reduced seizure-associated mortality. Histologic analysis of the spleens exposed to pilocarpine revealed altered white and red pulp anatomy and an increase in CD3+ T cells. Onset of status epilepticus (SE) and mortality were significantly decreased in perforin-deficient mice. Pilocarpine significantly increased spleen NK 1.1 and CD8+ cell percentage; in contrast, the brain inflammatory cell profile remained unchanged at the time of pilocarpine SE. BBB damage was reduced in the perforin-deficient pilocarpine-treated mice. SIGNIFICANCE: Immunosuppressant maneuvers such as splenectomy or lack of perforin decrease the onset or the severity of pilocarpine SE. Our results suggest that cytotoxic lymphocytes, and specifically the cytolytic factor perforin, may be key molecular players involved in the axis between peripheral intravascular inflammation and seizures.

S100B and S100B autoantibody as biomarkers for early detection of brain metastases in lung cancer.

BACKGROUND: S100B is an astrocytic protein that enters the blood stream when there is disruption of the blood-brain barrier (BBB). Over time, antibodies against S100B develop in the sera of patients who experience persistent or repeated BBB disruptions. We explored the use of serum S100B protein and S100B autoantibodies for the detection of brain metastasis in patients with lung cancer. METHODS: One hundred and twenty eight untreated patients with lung cancer who had brain imaging performed as part of their routine evaluation, participated. Serum S100B protein levels were measured by direct ELISA and S100B autoantibody levels by reverse ELISA. These levels in patients with brain metastases were compared alone and in combination to those without brain metastases. RESULTS: Eighteen (14%) patients had brain metastasis at the time of lung cancer diagnosis. An S100B cutoff of 0.058 ng/mL had a sensitivity of 89% and specificity of 43% for brain metastasis. When an autoantibody threshold of <2.00 absorbance units was used in conjunction with S100B, the sensitivity remained at 89%, and the specificity increased to 58%. The overall accuracy was 51% with S100B alone, improving to 62.5% when combined with autoantibodies. CONCLUSIONS: Serum S100B and S100B autoantibody levels may help to identify which lung cancer patients have brain metastases.

Is phosphorylated tau unique to chronic traumatic encephalopathy? Phosphorylated tau in epileptic brain and chronic traumatic encephalopathy.

Repetitive traumatic brain injury (rTBI) is one of the major risk factors for the abnormal deposition of phosphorylated tau (PT) in the brain and chronic traumatic encephalopathy (CTE). CTE and temporal lobe epilepsy (TLE) affect the limbic system, but no comparative studies on PT distribution in TLE and CTE are available. It is also unclear whether PT pathology results from repeated head hits (rTBI). These gaps prevent a thorough understanding of the pathogenesis and clinical significance of PT, limiting our ability to develop preventative and therapeutic interventions. We quantified PT in TLE and CTE to unveil whether a history of rTBI is a prerequisite for PT accumulation in the brain. Six postmortem CTE (mean 73.3 years) and age matched control samples were compared to 19 surgically resected TLE brain specimens (4 months-58 years; mean 27.6 years). No history of TBI was present in TLE or control; all CTE patients had a history of rTBI. TLE and CTE brain displayed increased levels of PT as revealed by immunohistochemistry. No age-dependent changes were noted, as PT was present as early as 4 months after birth. In TLE and CTE, cortical neurons, perivascular regions around penetrating pial vessels and meninges were immunopositive for PT; white matter tracts also displayed robust expression of extracellular PT organized in bundles parallel to venules. Microscopically, there were extensive tau-immunoreactive neuronal, astrocytic and degenerating neurites throughout the brain. In CTE perivascular tangles were most prominent. Overall, significant differences in staining intensities were found between CTE and control (P<0.01) but not between CTE and TLE (P=0.08). pS199 tau analysis showed that CTE had the most high molecular weight tangle-associated tau, whereas epileptic brain contained low molecular weight tau. Tau deposition may not be specific to rTBI since TLE recapitulated most of the pathological features of CTE.

Levels of S100B in brain and blood of rats with diabetic ketoacidosis.

Diabetic ketoacidosis (DKA) frequently causes subtle brain injuries in children. Rarely, these injuries can be severe and life threatening. The physiological processes leading to brain injury during DKA are poorly understood. S100B is a calcium-binding protein secreted by astrocytes. Elevated serum S100B levels are documented in several types of brain injuries. S100B may have either neuroprotective or neurotoxic effects, depending upon the concentration. We undertook the current studies to measure alterations in S100B production and secretion during DKA. We measured serum S100B concentrations in juvenile rats during and after DKA, and used immunohistochemistry to measure S100B expression in the hippocampus, cortex and striatum. Compared to levels in both normal and hyperglycemic control rats, serum S100B levels during DKA were significantly reduced. Serum S100B gradually rose after DKA, returning to levels of hyperglycemic controls by 72 h. S100B expression in the hippocampus was also significantly reduced 24h after DKA. There were no significant changes in S100B expression in other brain regions. Our findings contrast with those for other types of brain injuries in which both serum S100B levels and astrocyte S100B expression are typically elevated. These data suggest that serum S100B measurement cannot be used as an indicator of brain injury during DKA. Whether reduced S100B production or secretion is involved in the pathogenesis of DKA-related brain injury should be investigated.

Significance of ubiquitin carboxy-terminal hydrolase L1 elevations in athletes after sub-concussive head hits.

The impact of sub-concussive head hits (sub-CHIs) has been recently investigated in American football players, a population at risk for varying degrees of post-traumatic sequelae. Results show how sub-CHIs in athletes translate in serum as the appearance of reporters of blood-brain barrier disruption (BBBD), how the number and severity of sub-CHIs correlate with elevations of putative markers of brain injury is unknown. Serum brain injury markers such as UCH-L1 depend on BBBD. We investigated the effects of sub-CHIs in collegiate football players on markers of BBBD, markers of cerebrospinal fluid leakage (serum beta 2-transferrin) and markers of brain damage. Emergency room patients admitted for a clinically-diagnosed mild traumatic brain injury (mTBI) were used as positive controls. Healthy volunteers were used as negative controls. Specifically this study was designed to determine the use of UCH-L1 as an aid in the diagnosis of sub-concussive head injury in athletes. The extent and intensity of head impacts and serum values of S100B, UCH-L1, and beta-2 transferrin were measured pre- and post-game from 15 college football players who did not experience a concussion after a game. S100B was elevated in players experiencing the most sub-CHIs; UCH-L1 levels were also elevated but did not correlate with S100B or sub-CHIs. Beta-2 transferrin levels remained unchanged. No correlation between UCH-L1 levels and mTBI were measured in patients. Low levels of S100B were able to rule out mTBI and high S100B levels correlated with TBI severity. UCH-L1 did not display any interpretable change in football players or in individuals with mild TBI. The significance of UCH-L1 changes in sub-concussions or mTBI needs to be further elucidated.

Is peripheral immunity regulated by blood-brain barrier permeability changes?

S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses.

Consequences of repeated blood-brain barrier disruption in football players.

The acknowledgement of risks for traumatic brain injury in American football players has prompted studies for sideline concussion diagnosis and testing for neurological deficits. While concussions are recognized etiological factors for a spectrum of neurological sequelae, the consequences of sub-concussive events are unclear. We tested the hypothesis that blood-brain barrier disruption (BBBD) and the accompanying surge of the astrocytic protein S100B in blood may cause an immune response associated with production of auto-antibodies. We also wished to determine whether these events result in disrupted white matter on diffusion tensor imaging (DT) scans. Players from three college football teams were enrolled (total of 67 volunteers). None of the players experienced a concussion. Blood samples were collected before and after games (n = 57); the number of head hits in all players was monitored by movie review and post-game interviews. S100B serum levels and auto-antibodies against S100B were measured and correlated by direct and reverse immunoassays (n = 15 players; 5 games). A subset of players underwent DTI scans pre- and post-season and after a 6-month interval (n = 10). Cognitive and functional assessments were also performed. After a game, transient BBB damage measured by serum S100B was detected only in players experiencing the greatest number of sub-concussive head hits. Elevated levels of auto-antibodies against S100B were elevated only after repeated sub-concussive events characterized by BBBD. Serum levels of S100B auto-antibodies also predicted persistence of MRI-DTI abnormalities which in turn correlated with cognitive changes. Even in the absence of concussion, football players may experience repeated BBBD and serum surges of the potential auto-antigen S100B. The correlation of serum S100B, auto-antibodies and DTI changes support a link between repeated BBBD and future risk for cognitive changes.

The etiological role of blood-brain barrier dysfunction in seizure disorders.

A wind of change characterizes epilepsy research efforts. The traditional approach, based on a neurocentric view of seizure generation, promoted understanding of the neuronal mechanisms of seizures; this resulted in the development of potent anti-epileptic drugs (AEDs). The fact that a significant number of individuals with epilepsy still fail to respond to available AEDs restates the need for an alternative approach. Blood-brain barrier (BBB) dysfunction is an important etiological player in seizure disorders, and combination therapies utilizing an AED in conjunction with a "cerebrovascular" drug could be used to control seizures more effectively than AED therapy alone. The fact that the BBB plays an etiologic role in other neurological diseases will be discussed in the context of a more "holistic" approach to the patient with epilepsy, where comorbidity variables are also encompassed by drug therapy.

Blood-brain barrier P450 enzymes and multidrug transporters in drug resistance: a synergistic role in neurological diseases.

Drug penetration into the central nervous system (CNS) is controlled by the blood-brain barrier (BBB). Even though a number of strategies to circumvent the BBB and to improve drug access have been developed, drug resistance in CNS diseases remains an unmet clinical problem. We here review the mechanisms by which a healthy or pathological BBB influences drug distribution in the brain, with emphasis on the role of P450 metabolic enzymes and multi-drug transporter (MDT) proteins. In addition to the classic hepatic and gut biotransformation pathways, CNS expression of P450 enzymes may bear pharmacokinetic and pharmacodynamic significance exerting a metabolic activity and transforming parent drugs into specific products. We propose these mechanisms to play a major role in CNS drug resistant pathologies including refractory forms of epilepsy. Changes in the cerebrovascular hemodynamic conditions can affect expression of P450 enzymes and MDT proteins. This should be taken into account when developing in vitro experimental approaches to reproduce the physiological or pathological properties of the BBB. Finally, a link between P450 and MDT expression in the diseased brain and cell survival is discussed.

Efficacy of anti-inflammatory therapy in a model of acute seizures and in a population of pediatric drug resistant epileptics.

Targeting pro-inflammatory events to reduce seizures is gaining momentum. Experimentally, antagonism of inflammatory processes and of blood-brain barrier (BBB) damage has been demonstrated to be beneficial in reducing status epilepticus (SE). Clinically, a role of inflammation in the pathophysiology of drug resistant epilepsies is suspected. However, the use anti-inflammatory drug such as glucocorticosteroids (GCs) is limited to selected pediatric epileptic syndromes and spasms. Lack of animal data may be one of the reasons for the limited use of GCs in epilepsy. We evaluated the effect of the CG dexamethasone in reducing the onset and the severity of pilocarpine SE in rats. We assessed BBB integrity by measuring serum S100ß and Evans Blue brain extravasation. Electrophysiological monitoring and hematologic measurements (WBCs and IL-1ß) were performed. We reviewed the effect of add on dexamethasone treatment on a population of pediatric patients affected by drug resistant epilepsy. We excluded subjects affected by West, Landau-Kleffner or Lennox-Gastaut syndromes and Rasmussen encephalitis, known to respond to GCs or adrenocorticotropic hormone (ACTH). The effect of two additional GCs, methylprednisolone and hydrocortisone, was also reviewed in this population. When dexamethasone treatment preceded exposure to the convulsive agent pilocarpine, the number of rats developing status epilepticus (SE) was reduced. When SE developed, the time-to-onset was significantly delayed compared to pilocarpine alone and mortality associated with pilocarpine-SE was abolished. Dexamethasone significantly protected the BBB from damage. The clinical study included pediatric drug resistant epileptic subjects receiving add on GC treatments. Decreased seizure frequency (≥ 50%) or interruption of status epilepticus was observed in the majority of the subjects, regardless of the underlying pathology. Our experimental results point to a seizure-reducing effect of dexamethasone. The mechanism encompasses improvement of BBB integrity. Our results also suggest that add on GCs could be of efficacy in controlling pediatric drug resistant seizures.

Pathophysiological impact of cigarette smoke exposure on the cerebrovascular system with a focus on the blood-brain barrier: expanding the awareness of smoking toxicity in an underappreciated area.

Recent evidence has indicated that active and passive cigarette smoking are associated, in a dose-dependent manner, with dysfunction of normal endothelial physiology. Tobacco smoke (TS) may predispose individuals to atherogenic and thrombotic problems, significantly increasing the risk for ischemic manifestations such as acute coronary syndrome and stroke. Despite the strong evidence for an association between smoking and vascular impairment, the impact of TS exposure on the blood-brain barrier (BBB) has only been marginally addressed. This is a major problem given that the BBB is crucial in the maintenance of brain homeostasis. Recent data have also shown that chronic smokers have a higher incidence of small vessel ischemic disease (SVID), a pathological condition characterized by leaky brain microvessels and loss of BBB integrity. In the brain TS increases the risk of silent cerebral infarction (SCI) and stroke owing to the pro-coagulant and atherogenic effects of smoking. In this article we provide a detailed review and analysis of current knowledge of the pathophysiology of tobacco smoke toxicity at the cerebrovascular levels. We also discuss the potential toxicity of recently marketed "potential-reduced exposure products".