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  • We studied 22 new patients (Table 1) referred for evaluation to the Clinical Epilepsy Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NINDS/NIH; Bethesda, Maryland), or to the Department of Neurology Children's National Medical Center (CNMC; Washington, D. C.). Of these, 13 had surgery at NIH and nine had surgery at CNMC. Protocols for patient evaluation and surgical sample collection were approved by the NINDS and CNMC Intramural Clinical Research Committees. Clinical evaluation in each case included ictal video-EEG monitoring, 3-D–volumetric fast spin echo; and axial T1 and T2, coronal T2, and flair sequences performed on a 1.5 Tesla scanner (General Electric, http://www.gehealthcare.com). Mean age at seizure onset was 10.8 ± 10.7 y, and mean age at surgery was 21.4 ± 12.4 y. The 15 patients with MTLE had a significantly older age at surgery (27.4 y versus 14.9 y; p < 0.03) and a nonsignificant trend toward lower seizure onset age (7 y versus 12 y) than the seven patients without MTLE. Five patients with MTLE, but no patients without MTLE, had a history of febrile seizures (Table 1). All the patients with MTLE, compared with only one patient without MTLE who had a ganglioglioma, had clinical, electrographic, and/or imaging characteristics of MTLE [32,33]. We also studied one additional patient (patient 16) who presented with typical temporal lobe epilepsy but then developed a diffuse epileptic syndrome, resulting in a hemispherectomy. Table data removed from full text. Table identifier and caption: 10.1371/journal.pmed.0040180.t001 Clinical Information from NIH/CNMC Epilepsy Patient Cohort The patient 16 was an 8-y-old, right-handed boy with a history of febrile seizures presented with sudden onset of frequent complex partial and rare secondary generalized seizures, without clear etiology. Complex partial seizures were characterized were characterized by anxiety and repetition of a short verbal phrase, followed by staring and aphemia with preserved comprehension. Initial EEG showed left-sided slowing; MRI showed left temporal fullness and increased T2 signal. Cerebrospinal fluid revealed no red and three white cells, a protein of 13, and glucose of 77 mg/100 ml. Despite negative PCR for herpes, acyclovir was started. Seizures persisted despite eight antiepileptic drugs, steroids, IVIG, ganciclovir, and a vagal nerve stimulator. EEG showed left temporal periodic epileptiform discharges and left frontal and right sharp waves. A left mesial temporal lobectomy led to transient improvement, but within 2 mo seizure frequency approached baseline levels with semiology unchanged. MRI was unchanged, and there were no focal neurological findings. Invasive monitoring revealed left mid/posterior superior temporal gyrus seizure onset. After resection, frequent electrographic and clinical seizures arose from inferior and middle temporal gyrus and frontal lobe. After several additional focal resections, a hemispherectomy was performed. The patient has been seizure free without anticonvulsants for 1 y. He has a right hemiparesis with little finger function, mild receptive deficits, and more severe expressive deficits; language is improving. All patient surgical samples were reviewed by a clinical neuropathologist before submission for virological study. Samples from mesial temporal and neocortical resections and lateral temporal lobe resections were fixed in formalin and processed for standard pathological examination. All patients with MTS/MTLE showed variable degrees of neuronal loss and gliosis preferentially affecting the CA1 and CA3 areas, but also markedly affecting CA4, CA2, and the dentate gyrus in more severe cases. There was no evidence of inflammation or neuronal inclusions in any patient with MTS/MTLE. For isolation of PBMCs, blood samples were drawn into acid citrate dextrose solution A tubes (Becton Dickinson, http://www.bd.com) and were separated using lymphocyte separation medium (ICN Biomedicals, http://www.mpbio.com). Cells were stored in liquid nitrogen prior to DNA extraction. Serum samples were extracted within 4 h of collection. Fresh brain material was obtained during epilepsy brain resection. Brain tissue was stored on ice in Hibernate A medium, and astrocyte isolation was initiated within 30 min after obtaining tissue. DNA and RNA were isolated from fresh brain tissue using commercially available extraction kits according to manufacturers' instructions. The QIAamp blood kit was used for DNA extraction from PBMCs, the QIAamp Viral RNA kit was used for DNA extraction from serum (1.2 ml), and the DNeasy tissue kit (all kits from Qiagen, http://www.qiagen.com) was used for DNA extraction from fresh brain tissue. DNA and RNA were also extracted from cultured primary astroytes obtained from patient brain resections using DNeasy and RNeasy extraction kits (Qiagen). For RNA extraction from tissue, finely minced brain samples were resuspended in 350 μl QIAzol lysis reagent (Qiagen) and stored at −70 °C until use. RNA extraction was performed as per the manufacturer's directions using the RNeasy lipid tissue mini kit (Qiagen). Nested PCR for HHV-6 Major Capsid Protein: DNA amplification was performed, using nested primers specific for a highly conserved sequence corresponding to the major capsid protein gene (MCP) of HHV-6 [9,34]. The external primers amplified a 520 bp sequence, and the internal primers amplified a 258 bp sequence. PCR was performed using the Taq PCR master mix kit (Qiagen) as per manufacturer's instructions. DNA was amplified with 0.5 μM final primer concentration for 35 cycles using the following conditions: denaturation at 92 °C for 0.3 min, annealing at 55 °C for 0.3 min, and extension at 72 °C for 0.32 min. A total of 5 μl of primary PCR product was amplified using the internal primers with the same PCR conditions. A total of 10 μl of PCR product was subjected to electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. Viral DNA in patient samples was quantified using TaqMan PCR with primers specific for HHV-6A and HHV-6B as described previously [15,35]. The A- and B-specific primers are located within the immediate early region of HHV-6 and bind specifically to their respective variants. Control experiments demonstrated that A-specific primers amplified only the HHV-6A laboratory strains U1102 and GS, and that B-specific primers amplified the HHV-6B laboratory strain Z29 [15]. Standard and sample DNA were amplified in a 96-well reaction plate using the following conditions: 50 °C for 2 min for activation of uracil-N-glycosylase, 95 °C for 10 min to inactivate uracil-N-glycosylase, and 45 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (annealing and extension). All standards and samples were assayed in triplicate, and HHV-6 viral load was normalized to actin. Fresh tissue was obtained from epilepsy brain resections performed at NIH or CNMC. Tissue was kept on ice in Hibernate A medium (containing 2% B27 supplement, 0.5 mM glutamine, fungicide, and 1% penicillin/streptomycin). Meninges and blood vessels were removed, and tissue was minced into small pieces and dissociated in Earl balanced salt solution (containing 0.01% DNase, 20 U/ml papain, and 1:100 penicillin/streptomycin) in a 37 °C shaking water bath for 1 h. Digested tissue was mechanically dissociated with sterile pipettes and passed through a 60 μm filter. The single-cell suspension was centrifuged at 1,500 rpm for 10 min and separated on a Percoll gradient for 30 min at 15,000 rpm. The glial cell layer was isolated, washed, and plated overnight in poly-L-lysine coated flasks or two-well chamber slides in astrocytic medium (DMEM/F12 containing 10% FBS, 1% penicillin/streptomycin, and 0.01% gentamicin). Primary cultures were allowed to adhere overnight, and fresh medium was added the following day. After 3–4 wk in culture, astrocytes were stained for GFAP to determine culture purity. Primary astrocytes were isolated from patients with MTLE included in Table 2. Astrocytes from epilepsy patients 15 (Table 1) and patient 2a (previously described as patient 2 [15]) were grown on poly-L-lysine–coated chamber slides for 3–4 wk. Primary antibodies were prepared in PBS and consisted of 1:100 mouse anti-gp116/54/64 (Advanced Biotechnologies, http://www.abionline.com), 1:100 rabbit anti-GFAP (DAKO, http://www.dako.com), and 1:50 mouse anti-microglia marker CD68 (Santa Cruz Biotechnology, http://www.scbt.com). Slides were incubated with primary antibodies for 1 h at room temperature and then washed three times in PBS. Secondary antibodies conjugated to the appropriate fluorophore (Molecular Probes, http://probes.invitrogen.com) consisted of 1:100 anti-rabbit IgG FITC (green), 1:1,000 anti-mouse IgG rhodamine (red), and 1:100 anti-mouse IgG AMCA (blue). Slides were incubated with secondary antibody for 1 h at room temperature and washed three times in PBS. Where indicated, slides were counterstained with DAPI in mounting media (Molecular Probes). Mounted slides were visualized using a fluorescence microscope (Carl Zeiss, http://www.zeiss.com) at 20×, 32×, or 40× magnification. Table data removed from full text. Table identifier and caption: 10.1371/journal.pmed.0040180.t002 HHV-6 DNA Detection in Temporal Lobe Resections from NIH/CNMC epilepsy cohort (n=38) HHV-6 Infection of Primary Astrocytes: After 2–3 wk in culture, primary astrocytic cells from patient 20 were infected with HHV-6A and HHV-6B. Infection was performed as described previously [28]. Briefly, cultures were infected with freshly thawed cell-free supernatant from HHV-6A–infected JJahn or HHV-6B–infected SupT-1 cells at a ratio of 103 DNA viral copies/cell (quantified by TaqMan PCR). Mock infections were performed using culture medium from uninfected SupT1 or JJahn. Cultures were incubated for 3 h at 37 °C in 5% CO2, cultures were washed three times with PBS, and fresh medium was added. Cells were harvested for experiments 5 d after infection. Reverse Transcription-PCR and TaqMan for EAAT-2: RNA extracted from primary astrocytes was reverse transcribed using reagents from Applied Biosystems (http://www.appliedbiosystems.com) according to manufacturer's instructions. cDNA was amplified using primers specific for EAAT-2 [36]. PCR for EAAT-2 was run for 35 cycles using the following conditions: 95 °C for 45 s, 55 °C for 1 min, and 72 °C for 1 min. cDNA was also amplified using TaqMan primer/probe sequences specific for EAAT-2 and HPRT (synthesized from cDNA sequences by Synthegen, now Integrated DNA Technologies, http://www.idtdna.com). cDNA was amplified in a 96-well reaction plate using the following conditions: 50 °C for 2 min for activation of uracil-N-glycosylase, 95 °C for 10 min to inactivate uracil-N-glycosylase, and 45 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (annealing and extension).
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