bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐HT1Areceptors couple to activation of Akt, but not extracellular‐regulated kinase (ERK), in cultured hippocampal neurons
Authors: Cowen Daniel S. , Johnson‐Farley Nadine N. , Travkina Tatyana
Published: April 2005

5‐HT1Areceptors have been hypothesized to mediate some of the neuronal plasticity and behavioral responses stimulated by serotonin selective reuptake inhibitors. Although the cellular signaling pathways required for inducing these actions have not yet been determined, roles for the neuroprotective extracellular‐regulated kinase (ERK) mitogen‐activated protein (MAP) kinase and Akt pathways have been suggested. In the current studies we have utilized primary cultures to directly determine whether hippocampal 5‐HT1Areceptors couple to activation of Akt and ERK. We found that E18 hippocampal neurons exhibit a twofold activation of Akt when exposed to nanomolar concentrations of 5‐HT. The 5‐HT1/7receptor‐selective agonist 5‐carboxamidotryptamine maleate (5‐CT) and the 5‐HT1A/7receptor‐selective agonist 8‐hydroxy‐N,N‐dipropyl‐aminotetralin (8‐OH‐DPAT) maleate were found to activate Akt with equal efficacy, and similar potency, to 5‐HT. p‐MPPI and WAY‐100635, antagonists selective for 5‐HT1Areceptors, completely inhibited 5‐CT‐ stimulated Akt activation. Activation of Akt was also inhibited by pretreatment with pertussis toxin as well as the phosphatidylinositol 3‐kinase inhibitors, wortmannin and LY294002. In contrast, the 5‐HT selective antagonist, SB269970, caused no inhibition. Although the density of 5‐HT1Areceptors expressed by cultured neurons was sufficient to activate Akt, no activation of ERK was observed. These findings suggest that Akt, and not ERK, may be relevant to previous reports of hippocampal 5‐HT1Areceptors mediating neurotrophic responses.
Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture
Authors: Dolman Diana , Drndarski Svetlana , Abbott N. Joan , Rattray Marcus
Published: April 2005

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood–brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse‐transcriptase–polymerase chain reaction (RT–PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up‐regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood–brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood–brain barrier endothelium. Reverse‐transcriptase–PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non‐brain endothelia, is suppressed in the specialized endothelium of the blood–brain barrier.
A TECHNIQUE FOR ESTIMATING INCOME TRENDS FROM CURRENCY DATA AND AN APPLICATION TO NINETEENTH‐CENTURY BRAZIL
Authors: Leff Nathaniel H.
Published: April 2005

This paper develops a method for estimating long‐run trends in income growth from the data available on a country's currency stock. The method is applied to nineteenth‐centry Brazil. The results indicate that contrary to earlier beliefs, the country as a whole probably experienced only moderate growth in per‐capita income during the nineteenth century. The approach may also be useful for other countries where data shortages preclude estimates of national income by conventional methods.
A new method to calculate end‐member thermodynamic properties of minerals from their constituent polyhedra I: enthalpy, entropy and molar volume
Authors: VAN HINSBERG V. J. , VRIEND S. P. , SCHUMACHER J. C.
Published: April 2005

The thermodynamic properties of silicate minerals can be described as a linear combination of the fractional properties of their constituent polyhedra. In contrast, given the thermodynamic properties of these polyhedra, the thermodynamic properties of minerals can be estimated, where only the crystallography of the mineral needs to be known. Such estimates are especially powerful for hypothetical mineral end‐members or for minerals where experimental determination of their thermodynamic properties is difficult. In this contribution the fractional enthalpy, entropy and molar volume for 35 polyhedra have been determined using weighted multiple linear regression analysis on a data set of published mineral thermodynamic properties. The large number of polyhedra determined, allows calculation of a much larger variety of phases than was previously possible and the larger set of minerals used provides more confident fractional properties. The OH‐bearing minerals have been described by partial and total hydroxide coordinated components, which gives better results than previous models and precludes the need of a S–V term to improve estimates of entropy. However, the fractional thermodynamic properties only give adequate results for silicate minerals and double oxides, and should therefore not be used to estimate the properties of other minerals. The thermodynamic properties of ‘new’ minerals are calculated from a linear stoichiometric combination of their constituent polyhedra, resulting in estimates generally with associated uncertainty of <5%. The quality of such data appears to be of sufficient accuracy for thermodynamic modelling as shown for meta‐bauxites from the Alps and the Aegean, where the effect of Zn on the P–T stability of staurolite can be both qualitatively and quantitatively reproduced.
Neocortical and Hippocampal Changes after Multiple Pilocarpine‐induced Status Epilepticus in Rats
Authors: Valotta da Silva Alexandre , Regondi Maria Cristina , Cipelletti Barbara , Frassoni Carolina , Cavalheiro Esper Abrão , Spreafico Roberto
Published: April 2005

Summary: Purpose:Multiple episodes of pilocarpine‐induced status epilepticus (SE) in developing rats (P7–P9) lead to progressive epileptiform activity and severe cognitive impairment in adulthood. The present work studied possible underlying abnormalities in the neocortex and hippocampus of pilocarpine‐treated animals.

Methods:Wistar rats were submitted to pilocarpine‐induced SE at P7, P8, and P9, and were killed at P35. Immunocytochemistry was performed on 50‐μm vibratome sections, by using antibodies against nonphosphorylated neurofilament (SMI‐311), parvalbumin (PV), calbindin (CB), calretinin (CR), and glutamate decarboxylase (GAD‐65). Ten‐micron cryostat sections were processed for immunohistoblot by using antibodies against GluR1, GluR2/3, and GluR4 α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunits and NR2abN‐methyl‐d‐aspartate (NMDA) receptor subunit.

Results:Adult rats submitted to SE at P7–9 showed: (a) altered distribution of neocortical interneurons; (b) increased cortical and reduced hippocampal GAD‐65 expression; and (c) altered expression of hippocampal AMPA and NMDA receptors.

Conclusions:We conclude that multiple SE episodes during P7–9 generate long‐lasting disturbances that underlie behavioral and electrographic abnormalities later in life.
Time Course of Adverse Events in Patients with Localization‐related Epilepsy Receiving Topiramate Added to Carbamazepine
Authors: Majkowski Jerzy , Neto Walter , Wapenaar Robert , Van Oene Joop
Published: April 2005

Summary:  Purpose: To explore the time course of treatment‐emergent adverse events (AEs) during topiramate (TPM) adjunctive therapy.

Methods: Post hoc analyses were performed by using data from a large (264 subjects) multicenter, double‐blind, placebo‐controlled trial in which 200 mg/day TPM was added to carbamazepine (CBZ) with or without another antiepileptic drug (AED) in adults with treatment‐resistant partial‐onset seizures. The daily incidence and mean duration of the most common (≥5% incidence) AEs were calculated for patients completing the 12‐week study.

Results: The daily incidence of somnolence, headache, loss of appetite, nervousness, fatigue, dizziness, upper respiratory tract infection, and vertigo peaked during titration and declined to rates similar to that of placebo after the target TPM dose had been reached. In contrast, the daily incidence of paresthesia increased during titration and was maintained for the study duration. Relatively few patients had cognitive symptoms (9% with TPM, 5% with placebo), but these were the most common AEs associated with treatment discontinuation. Patient/investigator reports of weight loss increased gradually over the course of the trial, corresponding with the pattern of change in weight measured at study visits.

Conclusions: This study demonstrates that most of the more common AEs with TPM adjunctive therapy are transient. Patients can be counseled that most AEs emerging when TPM is initially added to CBZ can be expected to diminish with continued therapy.
Development of a Validated Clinical Case Definition of Generalized Tonic–Clonic Seizures for Use by Community‐based Health Care Providers
Authors: Anand Krishnan , Jain Satish , Paul Eldho , Srivastava Achal , Sahariah Sirazul A. , Kapoor Suresh K.
Published: April 2005

Summary:  Purpose: To develop and test a clinical case definition for identification of generalized tonic–clonic seizures (GTCSs) by community‐based health care providers.

Methods: To identify symptoms that can help identify GTCSs, patients with history of a jerky movements or rigidity in any part of the body ever in life were recruited from three sites: the community, secondary care hospital, and tertiary care hospital. These patients were administered a 14‐item structured interview schedule focusing on the circumstances surrounding the seizure. Subsequently, a neurologist examined each patient and, based on available investigations, classified them as GTCS or non‐GTCS cases. A logistic regression analysis was performed to select symptoms that were to be used for case definition of GTCSs. Validity parameters for the case definition at different cutoff points were calculated in another set of subjects.

Results: In total, 339 patients were enrolled in the first phase of the study. The tertiary care hospital contributed the maximal number of GTCS cases, whereas cases of non‐GTCS were mainly from the community. At the end of phase I, the questionnaire was shortened from 14 to eight questions based on statistical association and clinical judgment. After phase II, which was conducted among 170 subjects, three variables were found to be significantly related to the presence of GTCSs by logistic regression: absence of stress (13.1; 4.1–41.3), presence of frothing (13.7; 4.0–47.3), and occurrence in sleep (8.3; 2.0–34.9). As a case definition using only three variables did not provide sufficient specificity, three more variables were added based on univariate analysis of the data (incontinence during the episode and unconsciousness) and review of literature (injury during episode). A case definition consisting of giving one point to an affirmative answer for each of the six questions was tested. At a cutoff point of four, sensitivity was 56.9 (47.4–66.0) and specificity, 96.3 (86.2–99.4). Among the 197 GTCS and 26 new non‐GTCS patients recruited from hospitals from select SEAR Member Countries, in phase III, the sensitivity of this clinical case definition was 72% and specificity, 100%. A stratified analysis by gender in all the three phases did not show any differences between the sexes.

Conclusions: Based on these criteria, we recommend that all patients with a history of two or more episodes of jerking or rigidity of limbs, having a score of ≥4 in the case definition, be identified as having GTCSs and started on antiepileptic medications. This clinical case definition can be very useful for community‐based health care providers to identify and manage cases of GTCSs in the community. This should play a major role in the reduction of treatment gap for epilepsy in developing countries.
Depression and Anxiety Disorders in Pediatric Epilepsy
Authors: Caplan Rochelle , Siddarth Prabha , Gurbani Suresh , Hanson Rebecca , Sankar Ramen , Shields W. Donald
Published: April 2005

Summary: Purpose:This study examined affective disorders, anxiety disorders, and suicidality in children with epilepsy and their association with seizure‐related, cognitive, linguistic, family history, social competence, and demographic variables.

Methods:A structured psychiatric interview, mood self‐report scales, as well as cognitive and language testing were administered to 100 children with complex partial seizures (CPSs), 71 children with childhood absence epilepsy (CAE), and 93 normal children, aged 5 to 16 years. Parents provided behavioral information on each child through a structured psychiatric interview and behavior checklist.

Results:Significantly more patients had affective and anxiety disorder diagnoses (33%) as well as suicidal ideation (20%) than did the normal group, but none had made a suicide attempt. Anxiety disorder was the most frequent diagnosis among the patients with a diagnosis of affective or anxiety disorders, and combined affective/anxiety and disruptive disorder diagnoses, in those with suicidal ideation. Only 33% received some form of mental health service. Age, verbal IQ, school problems, and seizure type were related to the presence of a diagnosis of affective or anxiety disorder, and duration of illness, to suicidal ideation.

Conclusions:These findings together with the high rate of unmet mental health underscore the importance of early detection and treatment of anxiety disorders and suicidal ideation children with CPSs and CAE.
Deep EEG Recordings of the Mammillary Body in Epilepsy Patients
Authors: Van Rijckevorsel Kenou , Abu Serieh Basel , De Tourtchaninoff Marianne , Raftopoulos Christian
Published: April 2005

Summary:  Purpose: To our knowledge, the epileptic and nonepileptic electroencephalographic (EEG) discharges recorded within the human mammillary body (MB) and mammillothalamic tract (MTT) areas have never been published. Herein, we present the EEG recordings from these structures in patients with refractory epilepsy (RE).

Methods: Three men (ages 41–43 years) were enrolled in a clinical trial for deep brain stimulation (DBS) of MB‐MTT in RE. Previous evaluations had demonstrated a low likelihood of successful response to medication or resective surgery. DBS macroelectrodes were bilaterally implanted within the MB‐MTT under general anesthesia and their location checked by magnetic resonance imaging (MRI). We obtained a surface‐depth EEG for a 2‐ to 4‐day period, including monitoring of the cardiorespiratory and mnemonic functions.

Results: The background pattern of EEG recorded from MB‐MTT was low‐amplitude (usually <25 μv for MB and <20 μv for MTT) waves with a variable combination of theta–beta rhythms. In two patients, pseudoperiodic slow spikes were unilaterally recorded with or without clinical signs. For one patient, several focal ictal discharges were recorded in the right MB without scalp EEG changes.

Conclusions: The analysis of our depth EEG revealed that the theta–beta pattern represents the predominant physiologic profile of MB. Paroxysmal epileptiform discharges can be observed in human MB. These data supplement those available from animal observations.
Screening for Major Depression in Epilepsy with Common Self‐report Depression Inventories
Authors: Jones Jana E. , Hermann Bruce P. , Woodard John L. , Barry John J. , Gilliam Frank , Kanner Andres M. , Meador Kimford J.
Published: April 2005

Summary:  Purpose: Major depression is a common psychiatric comorbidity in chronic epilepsy that is frequently unrecognized and untreated. A variety of self‐report mood inventories are available, but their validity as well as ability to detect major depression in epilepsy remains uncertain. The purpose of this study was to determine the ability of two common depressive symptom inventories to identify major depression in people with epilepsy.

Methods: In total, 174 adult patients with epilepsy underwent standardized psychiatric interview techniques [Mini International Neuropsychiatric Interview (MINI) and Mood Disorders module of the Structured Clinical Interview for DSM‐IV Axis I Disorders–Research Version (SCID‐I)] to determine the presence of current major depression. Subjects completed two self‐report depression inventories [Beck Depression Inventory‐II (BDI‐II), Center for Epidemiological Study of Depression (CES‐D)]. The ability of these self‐report measures to identify major depression as identified by the gold standard structured interviews was examined by using diagnostic efficiency statistics.

Results: Both the BDI‐II and the CES‐D exhibited significant ability to identify major depression in epilepsy. All ROC analyses were highly significant (mean area under the curve, 0.92). Mean sensitivity (0.93) and specificity (0.81) were strong, with excellent negative predictive value (0.98) but lower positive predictive value (0.47).

Conclusions: Common self‐report depression measures can be used to screen for major depression in clinical settings. Use of these measures will assist in the clinical identification of patients with major depression so that treatment can be initiated.
Predictors for Negative Attitudes toward Subjects with Epilepsy: A Representative Survey in the General Public in Austria
Authors: Spatt Josef , Bauer Gerhard , Baumgartner Christoph , Feucht Martha , Graf Martin , Mamoli Bruno , Trinka Eugen ,   Austrian Section of the International League Against Epilepsy
Published: April 2005

Summary:  Purpose: To assess knowledge of and attitudes toward epilepsy in the Austrian general public and to identify independent predictors of negative attitudes.

Methods: A survey consisting of a representative sample of 2,128 Austrian adults was commissioned. A questionnaire similar to the ones used in earlier studies in other countries was used. The independent influence of socioeconomic variables and of knowledge and concepts about epilepsy on attitudes were analyzed by using logistic stepwise regression procedure.

Results: Nearly 10% of respondents expressed negative attitudes toward people with epilepsy. These figures are similar to those of other studies performed in comparable societies. In addition to being male and of low socioeconomic background, having little theoretical knowledge about epilepsy, misconceptions of epilepsy as a form of insanity, and no personal acquaintance with someone with epilepsy independently predispose to unfavorable attitudes toward epilepsy.

Conclusions: Information campaigns on epilepsy are likely to improve attitudes toward epilepsy when they target misconceptions about epilepsy and when they offer opportunities for personal acquaintance with people with epilepsy.
Levetiracetam Concentrations in Serum and in Breast Milk at Birth and during Lactation
Authors: Johannessen Svein I. , Helde Grethe , Brodtkorb Eylert
Published: April 2005

Summary:  Purpose: To study the pharmacokinetics of levetiracetam (LEV) at birth, during lactation, and in the nursed infant.

Methods: Eight consecutive breast‐feeding women with epilepsy treated with LEV twice daily and their infants were studied.

Results: The mean umbilical cord serum/maternal serum ratio was 1.14 (range, 0.97–1.45) (n = 4). The mean milk/maternal serum concentration ratio was 1.00 (range, 0.76–1.33) at 3 to 5 days after delivery (n = 7). At sampling 2 weeks to 10 months after delivery (n = 5), it was similar (range, 0.85–1.38). At 3 to 5 days after delivery, the infants had very low LEV serum concentrations (<10–15 μM), a finding that persisted during continued breast‐feeding. No malformations were detected, and in none of the infants did signs of adverse effects develop.

Conclusions: Our data indicate an extensive transfer of LEV from mother to fetus and into breast milk. However, breast‐fed infants had very low LEV serum concentrations, suggesting a rapid elimination of LEV.
Self‐reported Seizure Frequency and Time to First Event in the Seizure Monitoring Unit
Authors: Eisenman Lawrence N. , Attarian Hrayr , Fessler A. James , Vahle Victoria J. , Gilliam Frank
Published: April 2005

Summary:  Purpose: To compare seizure frequency reported in the clinic with time to first diagnostic event during video‐EEG monitoring. The effect of the artificial environment of the monitoring unit on self‐reported seizure frequency was explored.

Methods: The 155 consecutive patients were seen in the Washington University Epilepsy Center and subsequently underwent video‐EEG monitoring during 2001. Of these, 112 had a diagnostic event during monitoring; 31 left without having a definite event; and 12 could not provide an estimate of seizure frequency in the clinic. The time to first event was compared with self‐reported seizure frequency. The patients were then divided into three equal groups (tertiles) based on mean seizure frequency, and time to first seizure was compared between groups. Then the numbers of patients staying >7 days without ever having an event were compared between the low and high seizure‐frequency groups. Finally, Kaplan–Meier survival curves were calculated.

Results: No correlation was found between self‐reported seizure rate and time to diagnostic event (r= 0.18; p = 0.06). Time to first event was 2.8 days in the low seizure‐frequency group (mean, 2.2/month), 2.1 days in the medium (mean, 8.8/month), and 2.1 days in the high (mean, 24.1/month) groups, which were not significantly different (p = 0.19). Of patients in the low‐frequency group, 79% had an event within 7 days.

Conclusions: In the artificial environment of the monitoring unit, self‐reported outpatient seizure frequency is not an accurate predictor of duration of video‐EEG monitoring required to make a definitive classification of clinical events and should not contribute to the decision as to whether to refer a patient for monitoring.
Hematologic and lymphocyte immunophenotypic reference values for normal rhesus monkey (Macaca mulatta) umbilical cord blood; gravidity may play a role in study design
Authors: Rogers Linda B. , Kaack M. Bernice , Henson Michael C. , Rasmussen Terri , Henson Elizabeth , Veazey Ronald S. , Krogstad Donald J. , Davison Billie B.
Published: April 2005

Abstract:  Hematology and flow cytometry reference values for rhesus umbilical cord blood (UCB) were established in 17 healthy infant rhesus monkeys delivered by elective cesarean section 10 days preterm. The infants were born to age matched, singly caged primigravid or secundigravid dams. The hematology and flow cytometry values were determined by automated cell counter and by FACS. No significant differences were observed with respect to infant gender. With respect to gravida, the primigravid infants had a significantly higher percentage (P= 0.05) of CD20+ B lymphocytes in UCB.

These results provide useful reference values for future studies of maternal – fetal disease transmission, vaccine and drug evaluation in non‐human primate pregnancy, as well as fetal programming and immune modulation, gene therapy and the use of UCB as a source of stem cells for research and transplantation. Importantly, our results suggest that maternal gravidity may be an important variable to consider.
Variant Upstream Regulatory Region Sequences Differentially Regulate Human Papillomavirus Type 16 DNA Replication throughout the Viral Life Cycle
Authors: Hubert Walter G.
Published: April 2005

While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P
97
activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.
Identification of
cis
-Acting Elements That Mediate the Replication and Maintenance of Human Papillomavirus Type 16 Genomes in
Saccharomyces cerevisiae
Authors: Kim Kitai , Angeletti Peter C. , Hassebroek Elizabeth C. , Lambert Paul F.
Published: April 2005

Papillomaviruses contain small double-stranded DNA genomes that are maintained in persistently infected mammalian host epithelia as nuclear plasmids and rely upon the host replication machinery for replication. Papillomaviruses encode a DNA helicase, E1, which can specifically bind to the viral genome and support DNA synthesis. Under some conditions in mammalian cells, E1 is not required for viral DNA synthesis, leading to the hypothesis that papillomavirus DNA can be replicated solely by the host replication machinery. This machinery is highly conserved among eukaryotes. We and others found that papillomavirus DNA could replicate in a simple eukaryote,
Saccharomyces cerevisiae
. Specifically, papillomavirus DNA could substitute for the function of the autonomously replicating sequence (ARS) and centromere (CEN) elements that are normally both required for the stable replication of extrachromosomal DNAs in yeast. Furthermore, this form of replication in yeast was E1 independent. In this study, we map the elements in the human papillomavirus type 16 (HPV16) genome that can substitute for yeast ARS and CEN elements. A single element, termed
rep
, was identified that can substitute for ARS, and multiple elements, termed
mtc
, could substitute for CEN. The location of one of these
mtc
elements overlaps the location of
rep
, and this approximately 1,000-bp region of HPV16 was sufficient to support stable replication of a bacterial-yeast shuttle plasmid deleted of both ARS and CEN elements.
Characterization of the Interaction of Lassa Fever Virus with Its Cellular Receptor α-Dystroglycan
Authors: Kunz Stefan , Rojek Jillian M. , Perez Mar , Spiropoulou Christina F. , Oldstone Michael B. A.
Published: April 2005

The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as α-dystroglycan (α-DG), a cell surface receptor that provides a molecular link between the extracellular matrix and the actin-based cytoskeleton. In the present study, we show that LFV binds to α-DG with high affinity in the low-nanomolar range. Recombinant vesicular stomatitis virus pseudotyped with LFV glycoprotein (GP) adopted the receptor binding characteristics of LFV and depended on α-DG for infection of cells. Mapping of the binding site of LFV on α-DG revealed that LFV binding required the same domains of α-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin α1 and α2 chains for α-DG binding. Together with our previous studies on receptor binding of the prototypic immunosuppressive LCMV isolate LCMV clone 13, these findings indicate a high degree of conservation in the receptor binding characteristics between the highly human-pathogenic LFV and murine-immunosuppressive LCMV isolates.
Targeting Human Glioblastoma Cells: Comparison of Nine Viruses with Oncolytic Potential
Authors: Wollmann Guido , Tattersall Peter , van den Pol Anthony N.
Published: April 2005

Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.
Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines
Authors: Brown Bruce K. , Darden Janice M. , Tovanabutra Sodsai , Oblander Tamara , Frost Julie , Sanders-Buell Eric , de Souza Mark S. , Birx Deborah L. , McCutchan Francine E. , Polonis Victoria R.
Published: April 2005

A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.
Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins
Authors: Bertolotti-Ciarlet Andrea , Smith Jonathan , Strecker Karin , Paragas Jason , Altamura Louis A. , McFalls Jeanne M. , Frias-Stäheli Natalia , García-Sastre Adolfo , Schmaljohn Connie S. , Doms Robert W.
Published: April 2005

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus
Nairovirus
of the family
Bunyaviridae
, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G
N
and G
C
. To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G
N
and G
C
. Using these MAbs, we found that G
N
predominantly colocalized with a Golgi marker when expressed alone or with G
C
, while G
C
was transported to the Golgi apparatus only in the presence of G
N
. Both proteins remained endo-β-
N
-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the
cis
Golgi apparatus. Golgi targeting information partly resides within the G
N
ectodomain, because a soluble version of G
N
lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G
N
and G
C
also resulted in localization of soluble G
C
to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G
N
precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G
N
when it was expressed alone but were dispensable when G
C
was coexpressed. In neutralization assays on SW-13 cells, MAbs to G
C
, but not to G
N
, prevented CCHFV infection. However, only a subset of G
C
MAbs protected mice in passive-immunization experiments, while some nonneutralizing G
N
MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G
C
.
The Polypyrimidine Tract Binding Protein Is Required for Efficient Picornavirus Gene Expression and Propagation
Authors: Florez Paola M. , Sessions October M. , Wagner Eric J. , Gromeier Matthias , Garcia-Blanco Mariano A.
Published: April 2005

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that
trans
-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES
trans
-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.
Carboxyl-Proximal Regions of Reovirus Nonstructural Protein μNS Necessary and Sufficient for Forming Factory-Like Inclusions
Authors: Broering Teresa J. , Arnold Michelle M. , Miller Cathy L. , Hurt Jessica A. , Joyce Patricia L. , Nibert Max L.
Published: April 2005

Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein μNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of μNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue μNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of μNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of μNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in μNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with
Aequorea victoria
green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across μNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of μNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells.
Capsid Protein Synthesis from Replicating RNA Directs Specific Packaging of the Genome of a Multipartite, Positive-Strand RNA Virus
Authors: Venter P. Arno , Krishna Neel K. , Schneemann Anette
Published: April 2005

Flock house virus
(FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in
trans
from nonreplicating RNA. However, capsid protein synthesized in
cis
from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.
Induction of Long-Term Protective Antiviral Endogenous Immune Response by Short Neutralizing Monoclonal Antibody Treatment
Authors: Gros Laurent , Dreja Hanna , Fiser Anne Laure , Plays Marc , Pelegrin Mireia , Piechaczyk Marc
Published: April 2005

Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCasEretrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.
Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells
Authors: Campbell Stephanie A. , Lin Jennifer , Dobrikova Elena Y. , Gromeier Matthias
Published: April 2005

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.
Distinct Mechanisms of CD4+and CD8+T-Cell Activation and Bystander Apoptosis Induced by Human Immunodeficiency Virus Type 1 Virions
Authors: Holm Geoffrey H. , Gabuzda Dana
Published: April 2005

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4+and CD8+T cells. Infection of primary T-cell cultures with ELI6 induced CD4+T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4+and CD8+T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4+T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8+T cells was triggered by a soluble factor(s) secreted by CD4+T cells. HIV-1 virions activated CD4+and CD8+T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25+HLA-DR+T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4+and CD8+T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.
An Adenovirus Type 5 (Ad5) Amplicon-Based Packaging Cell Line for Production of High-Capacity Helper-Independent ΔE1-E2-E3-E4 Ad5 Vectors
Authors: Catalucci Daniele , Sporeno Elisabetta , Cirillo Agostino , Ciliberto Gennaro , Nicosia Alfredo , Colloca Stefano
Published: April 2005

Production of multiply deleted adenoviral (Ad) vectors with increased cloning capacity and reduced immunogenicity to adenovirus gene products requires the concomitant generation of efficient packaging cell lines. High expression levels of the complementing genes must be achieved in a coordinated fashion with viral replication. This is a particularly difficult task in light of the significant cytotoxicity displayed by adenoviral proteins. To this end, we developed a novel adenovirus-based amplicon with an Epstein-Barr virus origin of replication, Ad type 5 (Ad5) inverted terminal repeats, all Ad5 early region 2 (E2) genes, and the early region 4 (E4) open reading frame 6 (ORF6) under the control of a tetracycline-dependent promoter. The amplicon (pE2) was stably maintained in multiple copies in the nuclei of 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 (EBNA1) and allowed replication as a linear DNA upon induction of E2 and ORF6 gene expression. A stable cell line (2E2) was generated by introducing pE2 into 293EBNATet cells expressing the tetracycline-dependent transcriptional silencer and the reverse Tet transactivator (rtTA2). Upon induction with doxicycline, 2E2 cells produced higher levels of polymerase, precursor terminal protein (pTP), and DNA binding protein than noninduced 2E2 cells infected with first-generation Ad5 vector and supported efficient amplification of a multiply deleted Ad5 vector lacking E1, E2, E3, and E4 genes (Ad5ΔE
1-4
). The high cloning capacity of Ad5ΔE
1-4
(up to 12.6 kb) was exploited to construct a vector encoding the entire hepatitis C virus (HCV) polyprotein. Infection of HeLa cells by the resulting vector showed high levels of correctly processed HCV proteins.