Status epilepticus (SE) is one of the most serious manifestations of epilepsy. Systemic inflammation and damage of blood-brain barrier (BBB) are etiologic cofactors in the pathogenesis of pilocarpine SE while acute osmotic disruption of the BBB is sufficient to elicit seizures. Whether an inflammatory-vascular-BBB mechanism could apply to the lithium–pilocarpine model is unknown. LiCl facilitated seizures induced by low-dose pilocarpine by activation of circulating T-lymphocytes and mononuclear cells. Serum IL-1β levels increased and BBB damage occurred concurrently to increased theta EEG activity. These events occurred prior to SE induced by cholinergic exposure. SE was elicited by lithium and pilocarpine irrespective of their sequence of administration supporting a common pathogenetic mechanism. Since IL-1β is an etiologic trigger for BBB breakdown and its serum elevation occurs before onset of SE early after LiCl and pilocarpine injections, we tested the hypothesis that intravenous administration of IL-1 receptor antagonists (IL-1ra) may prevent pilocarpine-induced seizures. Animals pre-treated with IL-1ra exhibited significant reduction of SE onset and of BBB damage. Our data support the concept of targeting systemic inflammation and BBB for the prevention of status epilepticus.

It has long been held that chronic seizures cause blood-brain barrier (BBB) damage. Recent studies have also demonstrated that BBB damage triggers seizures. We have used the BBB osmotic disruption procedure (BBBD) to examine the correlation between BBB opening, pattern of white blood cells (WBCs) entry into the brain and seizure occurrence. These findings were compared to results from resected epileptic brain tissue from temporal lobe epilepsy (TLE) patients.We confirmed that a successful BBB osmotic opening (BBBD) leads to the occurrence of acute epileptiform discharges. Electroencephalography (EEG) and time-joint frequency analysis reveal EEG slowing followed by an increase in the 10-20 Hz frequency range. Using green fluorescent protein (GFP)-labeled WBCs (GFP-WBCs) suspended in Evans Blue we found that, at time of BBB-induced epileptiform discharges, WBCs populated the perivascular space of a leaky BBB. Similar results were obtained at time of pilocarpine seizure. No frank WBCs extravasation in the brain parenchyma was observed.In TLE brain specimens, CD45-positive leukocytes were detected only in the vascular and perivascular spaces while albumin and IgG extravasates were parenchymal. The pattern was similar to those observed in rats.Our data suggest that neither acute-induced nor chronic seizures correlate with WBC brain parenchymal migration while albumin and IgG brain leakage is a hallmark of acute and chronic seizures.

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations (''two-hit'' cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Twohit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal-epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.angiofibromas ͉ paracrine ͉ periungual fibromas ͉ TSC1 ͉ TSC2

Rationale: Lymphangioleiomyomatosis (LAM), occurring sporadically (S-LAM) or in patients with tuberous sclerosis complex (TSC), results from abnormal proliferation of LAM cells exhibiting mutations or loss of heterozygosity (LOH) of the TSC genes, TSC1 or TSC2. Objectives: To identify molecular markers useful for isolating LAM cells from body fluids and determine the frequency of TSC1 or TSC2 LOH. Methods: Candidate cell surface markers were identified using gene microarray analysis of human TSC2 2/2 cells. Cells from bronchoalveolar lavage fluid (BALF), urine, chylous effusions, and blood were sorted based on reactivity with antibodies against these proteins (e.g., CD9, CD44v6) and analyzed for LOH using TSC1-and TSC2-related microsatellite markers and single nucleotide polymorphisms in the TSC2 gene. Measurements and Main Results: CD44v6 1 CD9 1 cells from BALF, urine, and chyle showed TSC2 LOH in 80%, 69%, and 50% of patient samples, respectively. LAM cells with TSC2 LOH were detected in more than 90% of blood samples. LAM cells from different body fluids of the same patients showed, in most cases, identical LOH patterns, that is, loss of alleles at the same microsatellite loci. In a few patients with S-LAM, LAM cells from different body fluids differed in LOH patterns. No patients with S-LAM with TSC1 LOH were identified, suggesting that TSC2 abnormalities are responsible for the vast majority of S-LAM cases and that TSC1-disease may be subclinical. Conclusions: Our data support a common genetic origin of LAM cells in most patients with S-LAM, consistent with a metastatic model. In some cases, however, there was evidence for genetic heterogeneity between LAM cells in different sites or within a site.

ADP-ribosylation factors (ARFs) are critical in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)1 and BIG2 activate ARFs by accelerating replacement of bound GDP with GTP. Additional and differing functions of these Ϸ200-kDa proteins are now being recognized, as are their independent intracellular movements. Here, we describe the localization in COS7 cells by immunofluorescence microscopy of BIG2, but not BIG1, with structures that have characteristics of recycling endosomes during transferrin (Tfn) uptake and Tfn receptor (TfnR) recycling. Cell content of BIG2 and Rab11, but not TfnR, BIG1, Rab4, or Exo70, was increased after 60 min of Tfn uptake. BIG2, but not BIG1, appeared in density-gradient fractions containing TfnR, Rab11, and Exo70 after 60 min of Tfn uptake. Treatment of cells with BIG2 small interfering RNA (siRNA), but not BIG1 or control siRNAs, decreased BIG2 protein >90% without affecting BIG1, ARF, or actin content, whereas TfnR was significantly increased as was its accumulation in perinuclear recycling endosomes. Tfn release appeared unaffected by BIG1 siRNA but was significantly slowed from cells treated with BIG2 siRNA alone or plus BIG1 siRNA. We suggest that BIG2 has an important role in Tfn uptake and TfnR recycling, perhaps through its demonstrated interaction with Exo70 and the exocyst complex.ADP-ribosylation factor ͉ transferrin receptor ͉ Exo70 ͉ Rab11