Serum transthyretin monomer as a possible marker of blood-to-CSF barrier disruption.

The CNS is shielded from systemic influences by two separate barriers, the blood-brain barrier (BBB) and the blood-to-CSF barrier. Failure of either barrier bears profound significance in the etiology and diagnosis of several neurological diseases. Furthermore, selective opening of BBB tight junctions provides an opportunity for delivery of otherwise BBB impermeant drugs. Peripheral assessment of BBB opening can be achieved by detection in blood of brain-specific proteins that extravasate when these endothelial junctions are breached. We developed a proteomic approach to discover clusters of CNS-specific proteins with extravasation into serum that correlates with BBB openings. Protein profiles from blood samples obtained from patients undergoing iatrogenic BBB disruption (BBBD) with intra-arterial hyperosmotic mannitol were compared with pre-BBB opening serum. A low molecular weight protein (14 kDa) identified by mass spectroscopy as transthyretin (TTR) consistently correlated with BBBD. Protein gel electrophoresis and immunodetection confirmed that TTR was indeed extravasated in its monomeric form when CNS barriers were breached. The time course of TTR extravasation was compared with release from the brain of another BBB integrity marker, S-100beta (11 kDa). Kinetic analysis revealed that the appearance of S-100beta, presumably originating from perivascular astrocytic end feet, preceded extravasation of TTR by several minutes. Because TTR is localized primarily in choroid plexus and, as a soluble monomer, in CSF, we concluded that although S-100beta is a marker of BBB integrity, TTR instead may be a peripheral tracer of blood-to-cerebrospinal barrier.

RLIP76, a non-ABC transporter, and drug resistance in epilepsy.

BACKGROUND: Permeability of the blood-brain barrier is one of the factors determining the bioavailability of therapeutic drugs and resistance to chemically different antiepileptic drugs is a consequence of decreased intracerebral accumulation. The ABC transporters, particularly P-glycoprotein, are known to play a role in antiepileptic drug extrusion, but are not by themselves sufficient to fully explain the phenomenon of drug-resistant epilepsy. Proteomic analyses of membrane protein differentially expressed in epileptic foci brain tissue revealed the frequently increased expression of RLIP76/RALBP1, a recently described non-ABC multi-specific transporter. Because of a significant overlap in substrates between P-glycoprotein and RLIP76, present studies were carried out to determine the potential role of RLIP76 in AED transport in the brain. RESULTS: RLIP76 was expressed in brain tissue, preferentially in the lumenal surface of endothelial cell membranes. The expression was most prominent in blood brain barrier tissue from excised epileptic foci. Saturable, energy-dependent, anti-gradient transport of both phenytoin and carbamazepine were demonstrated using recombinant RLIP76 reconstituted into artificial membrane liposomes. Immunotitration studies of transport activity in crude membrane vesicles prepared from whole-brain tissue endothelium showed that RLIP76 represented the dominant transport mechanism for both drugs. RLIP76-/- knockout mice exhibited dramatic toxicity upon phenytoin administration due to decreased drug extrusion mechanisms at the blood-brain barrier. CONCLUSION: We conclude that RLIP76 is the predominant transporter of AED in the blood brain barrier, and that it may be a transporter involved in mechanisms of drug-resistant epilepsy.

Does Systemic Inflammation Play a Role in Pediatric Psychosis?

CONTEXT: Human and animal studies have suggested an underlying inflammatory mechanism for a variety of neuropsychiatric disorders, including schizophrenia. To date, most available reports focused on adult patients. OBJECTIVE: We wished to test the hypothesis that the first psychotic episode in youth is associated with inflammation. PATIENTS: We studied patients admitted to a pediatric inpatient psychiatric unit. Patients (n=80) had new-onset psychosis diagnosed using DSM-IV TR criteria for Psychosis NOS, Schizophreniform Disorder or Schizoaffective Disorder. Patients were matched for age, race and gender with inpatient controls without psychosis within the same unit (n=66). We also compared these values to normal pediatric hematologic values. To study the role of inflammation in youth with psychosis, we collected serum samples of 28 children presenting with first-episode psychosis and compared their serum cytokine and S100B levels to eight healthy controls. MAIN OUTCOME MEASURES: In this study, we measured serum markers of systemic inflammation. RESULTS: Leukocyte counts revealed a statistically significant increase in absolute monocytes compared to patients without psychosis (0.61 ± 0.282 k/ml vs. 0.496 ± 0.14 k/ml; p<0.01) and lymphocytes (2.51 ± 0.84 k/ml vs. 2.24 ± 0.72 k/ml; p<0.05) in patients with psychosis. All other hematologic values were similar between the groups. In addition, psychosis was characterized by increased serum levels of S100B, a peripheral marker of blood-brain barrier (BBB) damage. Several inflammatory mediators (e.g., TNF-α, IL-1ß, IL-6, IL-5, IL-10, and IFN-γ) were elevated in children with psychosis. CONCLUSIONS: These results strongly support a link between systemic inflammation, blood-brain barrier disruption and first-episode psychosis in pediatric patients.

Peripheral markers of brain damage and blood-brain barrier dysfunction.

PURPOSE: Occurrence of brain damage is frequently associated with abnormal blood-brain barrier (BBB) function. Two brain-specific proteins, S100beta and neuron-specific enolase (NSE) are released systemically in a variety of neurological diseases, but S100beta levels sometimes rise in the absence of neuronal damage, suggesting that S100beta is a marker of BBB rather than neuronal damage. METHODS: We measured both proteins in the serum of patients undergoing iatrogenic BBB disruption with intrarterial mannitol, followed by chemotherapy. RESULTS: Serum S100beta increased significantly after mannitol infusion (p<0.05) while NSE did not. Furthermore, in a model of intracerebral hemorrhage, S100beta increases in CSF did not lead to serum changes at a time when the BBB was intact. Modeling of S100beta release from the CNS suggested that low (<0.34 ng/ml) serum levels of S100beta are consistent with BBB opening without CNS damage, while larger increases imply synthesis and release from presumable damaged glia. CONCLUSIONS: Thus, S100beta in serum is an early marker of BBB openings that may precede neuronal damage and may influence therapeutic strategies. Secondary, massive elevations in S100beta are indicators of prior brain damage and bear clinical significance as predictors of poor outcome or diagnostic means to differentiate extensive damage from minor, transient impairment.

Small vessel ischemic disease of the brain and brain metastases in lung cancer patients.

BACKGROUND: Brain metastases occur commonly in patients with lung cancer. Small vessel ischemic disease is frequently found when imaging the brain to detect metastases. We aimed to determine if the presence of small vessel ischemic disease (SVID) of the brain is protective against the development of brain metastases in lung cancer patients. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective cohort of 523 patients with biopsy confirmed lung cancer who had received magnetic resonance imaging of the brain as part of their standard initial staging evaluation was reviewed. Information collected included demographics, comorbidities, details of the lung cancer, and the presence of SVID of the brain. A portion of the cohort had the degree of SVID graded. The primary outcome measure was the portion of study subjects with and without SVID of the brain who had evidence of brain metastases at the time of initial staging of their lung cancer.109 patients (20.8%) had evidence of brain metastases at presentation and 345 (66.0%) had evidence of SVID. 13.9% of those with SVID and 34.3% of those without SVID presented with brain metastases (p<0.0001). In a model including age, diabetes mellitus, hypertension, hyperlipidemia, and tobacco use, SVID of the brain was found to be the only protective factor against the development of brain metastases, with an OR of 0.31 (0.20, 0.48; p<0.001). The grade of SVID was higher in those without brain metastases. CONCLUSIONS/SIGNIFICANCE: These findings suggest that vascular changes in the brain are protective against the development of brain metastases in lung cancer patients.

Bone marrow-derived cells are the major source of MMP-9 contributing to blood-brain barrier dysfunction and infarct formation after ischemic stroke in mice.

Matrix metalloproteinase (MMP)-9 has been shown to contribute to blood-brain barrier (BBB) disruption, infarct formation, and hemorrhagic transformation after ischemic stroke. The cellular source of MMP-9 detectable in the ischemic brain remains controversial since extracellular molecules in the brain may be derived from blood. We here demonstrate that bone marrow-derived cells are the major source of MMP-9 in the ischemic brain. We made bone marrow chimeric mice with MMP-9 null and wild-type as donor and recipient. After 90 min of transient focal cerebral ischemia, MMP-9 null mice receiving wild-type bone marrow showed comparable outcomes to wild-type in brain MMP-9 levels and BBB disruption (endogenous albumin extravasation) at 1 h post-reperfusion and infarct size at 24 h post-reperfusion. In contrast, wild-type animals replaced with MMP-9 null bone marrow showed barely detectable levels of MMP-9 in the ischemic brain, with attenuations in BBB disruption and infarct size. MMP-9 null mice receiving wild-type bone marrow showed enhanced Evans blue extravasation as early as 1 h post-reperfusion compared to wild-type mice replaced with MMP-9 null bone marrow. These findings suggest that MMP-9 released from bone marrow-derived cells influences the progression of BBB disruption in the ischemic brain.

Loss of flow induces leukocyte-mediated MMP/TIMP imbalance in dynamic in vitro blood-brain barrier model: role of pro-inflammatory cytokines.

There is substantial evidence linking blood-brain barrier (BBB) failure during cerebral ischemia to matrix metalloproteinases (MMP). BBB function may be affected by loss of shear stress under normoxia/normoglycemia, as during cardiopulmonary bypass procedures. The present study used an in vitro flow-perfused BBB model to analyze the individual contributions of flow, cytokine levels, and circulating blood leukocytes on the release/activity of MMP-9, MMP-2, and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs), TIMP-1, and TIMP-2. The presence of circulating blood leukocytes under normoxic/normoglycemic flow cessation/reperfusion significantly increased the luminal levels of MMP-9 and activity of MMP-2, accompanied by partial reduction of TIMP-1, complete reduction of TIMP-2 and increased BBB permeability. These changes were not observed during constant flow with circulating blood leukocytes, or after normoxic/normoglycemic or hypoxic/hypoglycemic flow cessation/reperfusion without circulating blood leukocytes. The addition of anti-IL-6 or anti-TNF-alpha antibody in the lumen before reperfusion suppressed the levels of MMP-9 and activity of MMP-2, had no effect on TIMP-1, and completely restored TIMP-2 and BBB integrity. Injection of TIMP-2 in the lumen before reperfusion prevented the activation of MMP-2 and BBB permeability. These data indicate that blood leukocytes and loss of flow are major factors in the activation of MMP-2, and that cytokine-mediated differential regulation of TIMP-1 and TIMP-2 may contribute significantly to BBB failure.